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ARZNEIMITTEL FORSCHUNG DRUG RESEARCH Einteilung dieses Jahrgangs Subdivision of this annual volume Band I : Band I I :

1-908 909-1710

33. Jahrgang (1983) Heft 1-12 und Sonderausgaben la, 2a, 4a, 7a und 9a ARZNEIM.-FORSCH./DRUG RES. EDITIO CANTOR AULENDORF

F r o m the Department o f Clinical Chemistry and C l i n i c a l Biochemistry i n the Surgical C l i n i c o f the University of M u n i c h (FR Germany)

Biochemistry and Applications of Aprotinin, the Kallikrein Inhibitor from Bovine Organs 1

By H . F r i t z and G. Wunderer )

Summary: The basic proteinase inhibitor from bovine or­ gans, aprotinin (active ingredient of TrasyloP) has been ex­ tensively studied with respect to its chemical, physical and biochemical properties and its inhibitory mechanism of ac­ tion. It is widely used as a valuable tool for studying protein/ protein interactions and protein conformation at the mole­ cular level. There are numerous examples of the usefulness of aprotinin in biochemical and biomedical research. It has also become a valuable drug for the treatment of various diseases like, e.g. hyp er fibrinolytic hemorrhage and trauma­ tic-hemorrhagic shock. The purpose of this paper is threefold. It summarizes our present knowledge of the subject in various disciplines; it provides the active scientist with basic data for his experi­ mental work; and above all it points the way to future direc­ tions of aprotinin research. Zusammenfassung: Biochemie krein-Inhibitors Aprotinin aus

Key words: Aprotinin,

und Anwendung Rinderorganen

biochemistry,

mechanism

des

Kalli­

of action · Kallikrein,

Contents 1. Introduction: Discovery, distribution and isolation 2. Structure and physicochemical Properties 2.1. Structural characteristics 2.2. Stability, optical data and behavior in dialysis 3. Inhibitory characteristics and biological aspects 3.1. Units and assays 3.2. Inhibitory specificity and biological aspects 3.3. Inhibition mechanism and kinetics constants 4. Molecular mechanism of inhibition 4.1. Formation and structure of aprotinin-proteinase complexes 4.2. Analogs of aprotinin formed by enzymatic and chemical modification 4.3. The structural basis of kallikrein inhibition 5. Special properties relevant to administration of aprotinin 5.1. Interactions with glycoproteins and mucopolysaccharides 5.2. Immunogenicity 5.3. Elimination after administration 5.4. Effects on cellular function 6. Biochemical and biomedical applications 6.1. General applications 6.1.1. Isolation of proteinases 6.1.2. Crystallization of proteinases 6.1.3. Use as molecular weight marker 6.1.4. Identification of kallikrein activity

0 Present address: Dr. G. Wunderer, 1. Frauenklinik der Universität München (FR Germany). A r z n e i m . - F o r s c h . / Drug Res. 33 ( I ) , Nr. 4 (1983) Fritz et al. - Aprotinin

Der basische Proteinase-Inhibitor Aprotinin (Wirkstoff von TrasyloP) aus Rinderorganen wurde hinsichtlich seiner che­ mischen, physikalischen und biochemischen Eigenschaften sowie seiner Reaktionsweise mit Enzymen eingehend unter­ sucht. Aprotinin dient heute allgemein als wertvolles Agens zum Studium der Wechselwirkungen von Proteinen und der Protein-Konformation auf molekularer Basis. In zahlreichen Anwendungsbeispielen in der biochemischen und biomedi­ zinischen Forschung wurden mit Hilfe des Aprotinin rich­ tungsweisende Erkenntnisse gewonnen. Aprotinin hat sich auch als potenter Wirkstoff zur Behandlung verschiedener Krankheiten erwiesen, so ζ. B. bei hyperfibrinolytischen Blutungen und beim traumatisch-hämorrhagischen Schock. Der vorliegende Artikel dient einem dreifachen Zweck: Er faßt den gegenwärtigen Erkenntnisstand in den verschiedenen Fachdisziplinen zusammen, er bietet dem experimentell tätigen Wissenschaftler Basisdaten für seine Versuchsvorhaben und er gibt Hinweise für neue Zielsetzungen, die mittels Aprotinin angegangen werden können.

6.1.5. 6.1.6. 6.2. 6.3. 6.4. 6.5. 6.6. 6.7. 6.8. 6.9. 6.10. 6.11. 7. 7.1. 7.2. 8. 9. 9.1. 9.1.1. 9.1.2. 9.1.3. 9.1.4. 9.1.5. 9.1.6. 9.1.7. 9.2. 9.2.1. 9.2.2. 10.

inhibition

· Proteinase

inhibitors,

bovine -

TrasyloP

Prevention of proenzyme activation and/or proteolytic degradation Termination of limited proteolysis Tool for protein conformation and protein-protein interaction studies Tissue culture and organ preservation Inhibition of (transformed) cell growth Effects on leukocytes and macrophages Blood preservation and inhibition of platelet aggregation Wound healing Tool for studies of muscle metabolism and renal function Effects on fertilization Experimental shock Effect on virus replication Clinical use General aspects Interaction with plasmin and plasma kallikrein Synopsis Appendix on aprotinin applications General comments Availability Storage Dialysis, ultrafiltration and chromatography Interaction with heparin Immunoassays Inhibition studies Use in biological studies Inhibition assays Photometric assay Titrimetric assays References 479

1. Introduction: Discovery, distribution and isolation A p r o t i n i n ) was independently discovered by Kraut et al. [1] as a kallikrein " i n a c t i v a t o r " i n bovine l y m p h nodes, and by K u n i t z and N o r t h r o p [2] as a trypsin inhibitor i n the bovine pancreas. Later, Werle et al. also found kallikrein-inhibiting activities i n extracts o f other bovine organs, such as the lung, parotid gland, spleen, liver, pancreas, and seminal vesicles. Lower activities were found in the t h y r o i d gland, kidney and mucous membranes o f the trachea and esophagus, and i n extracts from the same organs o f sheep and goat [ 3 - 6 ] . M o r e recent investigations have shown that this kallikreininhibiting activity is due i n most, i f not a l l , cases to the presence o f aprotinin or aprotinin-like inhibitors i n these tissues [5-7]. In addition, aprotinin has been found in the following bovine organs or tissues: Ovary [8], heart, thyroid, posterior p i tuitary, and the rumen mucosa [9] as well as i n cartilage and aorta [10]. Remarkably, the two latter tissues are known to be highly resistant to tumor invasion. Recently, H o c h strasser and W ä c h t e r discovered aprotinin-like inhibitors as components o f the human and bovine inter-a-trypsin i n h i b i tor [11] and i n free form i n bovine serum [12]. O f special interest, i n view o f its biological function [13] is the occurrence o f aprotinin i n mast cells, detected by the i n direct immunofluorescence technique [13, 14], and its behavior as an intracellular compound [4, 5]. The mast cell o r i gin o f aprotinin is indicated also by a similar distribution pattern: organs known to contain high numbers o f these connective tissue cells are also rich i n aprotinin (especially lung, parotis and pancreas) and vice versa. Possibly, aprot i n i n is involved in the regulation o f mast cell proteinase activities [13]. 2

2. Structure and physicochemical properties 2.1. Structural characteristics A p r o t i n i n , a polypeptide w i t h a molecular weight o f 6,51 2, is obtainable i n crystalline form [2, 16]. It consists o f 58 amino acid residues that are arranged i n a single polypepti de chain. This chain is cross-linked by three disulfide bridges, one o f which (Cys-14-Cys-38) is readily split by reducing agents. The primary structure o f aprotinin is shown i n Fig. 1 [5, 17, 18]. Information on the three-dimensional structure was o b tained by X-ray crystallography, which revealed a p y r i f o r m molecule (Fig. 2) [19, 20]. The polypeptide chain is folded so that the hydrophobic radicals are concentrated i n the interior o f the molecule, whereas all o f the hydrophilic radicals, except for the side chain Asp-43, are on the outside which is exposed to the aqueous environment. This arrangement results in a very compact tertiary structure and is m a i n l y re-

For commercial purposes, aprotinin is isolated by classical methods such as fractional precipitation, gel filtration and ion exchange chromatography, being extracted solely from bovine lung, pancreas and parotid glands [5]. These tissues contain approx. 1500 K I U (= biological kallikrein units) (lung) or 400 K I U (pancreas, parotid gland) per gram wet weight [4, 5]. For the rapid purification o f aprotinin or aprotinin-like inhibitors o n a laboratory scale i n good yields (up to 90%), acidic extraction followed by affinity chromatography on water-insoluble enzyme derivatives (e.g. trypsinSepharose or trypsin-cellulose) is the method o f choice [8, 15]. O n the other hand, water-insoluble aprotinin derivatives are widely used for the isolation o f proteinases (see sect i o n 6.1.). 2

) Aprotinin is also known as bovine pancreatic trypsin inhibitor (Kunitz) (BPTI) and trypsin-kallikrein inhibitor (TKI); it is the active ingredient of Trasylol®, a drug registered in the name of Bayer Leverkusen (FR Germany). In some countries Kallikrein is a registered trademark of Bayer.

Fig. 1: Primary structure of aprotinin according to Kassell and Laskowski [17] and Anderer and Hörnle [18]. The inhibitor molecule consists of a single polypeptide chain of 58 amino acid residues cross-linked by 3 disulfide bridges. 480

Fig. 2: Tertiary structure of aprotinin according to Huber et al. [19] and Deisenhofer and Steigemann [20]. The molecule has a length of about 29 A, a diameter of 19 Ä, and contains a double-stranded antiparallel /?-sheet (from Ala-16 to Gly-36), twisted to form a right-handed double helix with 14 residues per turn. Amino acid residues included in the hatched area are in close contact with the enzyme in the trypsin-inhibitor complex. Residue number 19 is not included in this contact region, but its basic nature may be important for kallikrein inhibition in some cases; cf. Table 4 and Section 4.3. Arzneim.-Forsch./ Drug Res. 33 ( I ) , Nr. 4 (1983) Fritz et al. - Aprotinin

sponsible for the remarkable stability o f aprotinin against denaturation by high temperature, acids, alkalies and organ­ ic solvents or proteolytic degradation [21]. Other interesting features are: (i) T h e highly dipolar char­ acter o f a p r o t i n i n due to concentration o f the negatively charged radicals at one end o f the molecule, viz. the bottom of the pear (see Fig. 2); and (ii) the strong basicity of the aprotinin molecule, w i t h an isoelectric point close to 10.5 [21].

Table 1: Conversion factors between the various units or inhibitor units (IU) that are recommended or used to express the inhibitory activity of aprotinin. Units or inhibitor units are defined as the reduction in substrate turnover, ex­ pressed in μιηοΐ per time interval, caused by aprotinin as compared with an inhibitor-free control sample. Units given Units wanted

a

2.2. Stability, optical data and behaviour in dialysis A p r o t i n i n can be heated for a short time i n dilute acid at 100 C or i n 2.5% trichloroacetic acid at 80 °C without los­ ing any activity. It can be exposed to solutions with extreme pH-values ranging from 1 to 12.6; inactivation begins at p H 12.8 at r o o m temperature [21]. These data were obtained by various methods, such as i n h i b i t i o n studies [21], N M R (nu­ clear magnetic resonance), and R a m a n measurements [22]. A p r o t i n i n may be kept i n a salt or buffer solution, e.g. 0.15 mol/1 N a C l , at room temperature for at least 18 months w i t h o u t any loss o f activity. The i n h i b i t o r is also stable and soluble i n water, 70% (v/v) methanol, 70% (v/v) ethanol, and 50% (v/v) acetone [21]. Another outstanding property o f a p r o t i n i n is its uncommon stability to proteolytic degradation by other proteinases [7, 21]. So far, only thermolysin has been found capable of d i ­ gesting " n a t i v e " aprotinin after heat labilization at 6 0 - 8 0 °C [23]. In a 1 m g / m l solution of a p r o t i n i n , absorption of A = 0.84 occurs at 280 n m (1 cm light path), relatively independently o f p H [21]. I n the presence o f bactericidal agents, the i n h i b i ­ tor concentration should be optically determined at an alka­ line p H . The absorption m a x i m u m o f aprotinin is then shift­ ed from 277 n m at p H 7 (specific absorbance: 0.89 cmVmg) to 297 n m i n 0.1 Ν N a O H (specific absorbance: 1.32 cmVmg), thus differing from the absorption range o f the commonly used preservatives (Arens, Α., 1970, unpublished data). O w i n g to its low molecular weight and high basicity, apro­ t i n i n penetrates or adheres to the commonly employed dia­ lysis tubes. I t is advisable, therefore, to use either acetylated material [24] or ultrafiltration membranes with an exclusion l i m i t o f 5000 daltons. Small quantities o f aprotinin should be kept i n suitable salt solutions (e.g. i n volatile buffers) to avoid adsorption o f the inhibitor on negatively charged sur­ faces during dialysis or filtration through columns (e.g. Sephadex). e

3. Inhibitory characteristics and biological aspects 3.1. Units and assays A p r o t i n i n is often called a "broad-specificity i n h i b i t o r " as an expression o f its ability to i n h i b i t various proteinases such as those listed in Table 2. T h e " k a l l i k r e i n inactivator u n i t " ( K I U or K I E ) is i n worldwide use as a measure o f aprotinin activity and is accepted as such [ 3 - 7 ] . One K I U is defined as that amount o f aprotinin which decreases the activity o f two biological kallikrein units ( K U ) by 50%. (One K U is defined as that amount o f kallikrein which, when i n ­ jected intravenously, causes the same decrease i n dog carotid blood pressure as 5 m l of urine taken from 50 L , collected from healthy human individuals and dialyzed for 24 h against running tap water [3, 4]. This unit is also known as a Frey u n i t for F U [1, 3]). As the biological assay is tedious to perform and has a high standard deviation, enzymatic assays with synthetic sub­ strates are preferable. Compared w i t h kallikrein, inhibition of trypsin by aprotinin (expressed i n μτηοϊ o f substrate turn­ over inhibited in unit time) proceeds much more rapidly and the resulting titration curve is linear over nearly the whole range, i.e. up to 90% inhibition o f the applied amount o f enzyme. Therefore, trypsin inhibition tests are advanta­ geous and commonly used for the assay o f aprotinin. The Enzyme Commission of the FIP (Federation Internationale Pharmaceutique) recommends a titrimetric assay in which Arzneim.-Forsch. / Drug Res. 33 ( I ) , Nr. 4 (1983) Fritz et al. - Aprotinin

KIU ) F.l.P. units ) //kat ) b

c

IUßAPA

a b

c

d

a

KIU ) biol. assay

b

F.l.P. units ) (μπιοί BAEE/min)

c

^kat ) (//mol BAEE/s)

d

I U ) ΒΑΡΑ (μηιοί ΒΑΡΑ/min)

χ1

x30

χ 1800

x615

χ 1/30 χ 1/1 800 χ 1/615

χ 1 χ 1/60 χ 30/615 (= 1/20.5)

χ 60 χ 1 χ 1800/615 (=2.93)

χ 20.5 χ 1/2.93 1

) "Kallikrein inactivator unit" based on a biologic assay, see text. ) Units based on a titrimetric assay with BzArgOEt (BAEE) as (hog) trypsin substrate as recommended by the Enzyme Commission of the Federation Internationale Pharmaceutique (F.I.P.); see text. ) Units recommended by the NC-IUB (Nomenclature Committee of the In­ ternational Union of Biochemistry), Eur. J. Biochem. 97, 319 (1979). ) Inhibitor units based on a photometric assay with BzArgpNA (ΒΑΡΑ) as substrate and porcine trypsin as enzyme, see text.

a

residual trypsin activity is determined w i t h BzArgOEt ( N benzoyl-L-arginine ether ester, also B A E E for short) as the substrate under conditions specified by the I U B (Internation­ al U n i o n o f Biochemistry) [25, 26]. The conversion factors between the various recommended units are given i n Table 1. Highly purified aprotinin free o f water and salt ist found to have a specific activity o f 7,150 ± 200 K I U / m g or 0.14 //g/KIU. A photometric assay is also very useful for practical pur­ poses. I n this test, trypsin i n h i b i t i o n by aprotinin is deter­ mined by means o f B z A r g p N A (N*-benzoyl-arginine-pnitroanilide, also Β Α Ρ Α for short) as the substrate at 405 n m [21, 27]. The racemic substrate mixture D , L - B A P A (0.77 mmol/1) previously used i n this assay should be replaced by L - B A P A (0.383 mmol/1). Inhibition o f trypsin by the D form and solubility problems frequently encountered w i t h the mixture o f the two enantiomers are then avoided [28]. The biological units ( K I U ) can be calculated from the i n h i ­ bitor units that are obtained w i t h porcine trypsin and L B A P A as substrate by the equation: IUBAPA ( μ π ι ο ί / min) χ 615 = K I U (see Table 1). W i t h the photometric assay (substrate L - B A P A ) about 6 0 - 3 0 0 pmol aprotinin (in a given concentration o f approxi­ mately 0.6-3 yumol/l) and w i t h the titrimetric assay (sub­ strate BAEE) up to twice this amount or concentration o f aprotinin can be measured under the conditions given i n the Appendix. Whereas the photometric assay is especially suit­ able for determination o f aprotinin concentrations, the titrimetric assay is the method o f choice i f optimal condi­ tions for enzyme catalysis are required. Principally, trypsin inhibition by a p r o t i n i n may be assayed also w i t h more en­ zyme-specific chromatogenic ("chromogenic") or fluorogenic peptide substrates [29, 30]. However, due to the higher sensitivity (approx. 10- to lOOfold) reached w i t h such sub­ strates, complex formation may proceed more slowly and the range o f the linear part o f the titration curve may be smaller, both effects being o f disadvantage for practical purposes. The same holds true i f instead o f trypsin i n h i b i ­ tion tissue kallikrein i n h i b i t i o n is determined by means o f a chromogenic or fluorogenic peptide substrate, e.g. D ValLeuArg-p-nitroanilide [31] or ProPheArg-4-methyl-coumaryl-7-amide [32]. A kallikrein i n h i b i t i o n assay (preferably w i t h D-ValLeuArg-p-nitroanilide = S-2266 [31]) may be ad­ vantageous, however, i f aprotinin has to be quantified i n tis­ sue extracts or body fluids containing i n addition naturally occuring trypsin inhibitors. I n such cases the influence o f the extracts on kallikrein activity also has to be considered and control samples w i t h k n o w n aprotinin concentrations should be used as a reference standard ( M . Jochum and H . Fritz, manuscript i n preparation). Note, kallikrein i n h i b i t i o n alone 481

does not prove the presence o f aprotinin i n a test sample; for unequivocal identification o f aprotinin at least the i n h i b i t i o n ratio towards its major target enzymes (trypsin, chymotrypsin, plasmin, plasma kallikrein and tissue kallikrein; cf. T a ­ ble 2) has to be detected i f specific immunological methods are not available. In lieu o f synthetic substrates, use may be made of the na­ tural kallikrein substrates, the kininogens, for the assay o f aprotinin activity based on kallikrein or trypsin i n h i b i t i o n [33]. When plasmin inhibition is assayed as described pre­ viously [34], 1 antiplasmin unit ( A P U ) corresponds to 40 K I U o f aprotinin. A radioimmunoassay technique devel­ oped by Fink and Greene permits specific detection o f m i n ­ imal quantities o f aprotinin i n biological fluids or tissue ex­ tracts [35]. 3.2. Inhibitory specificity and biological aspects The most striking feature o f aprotinin is its broad inhibitory specificity. Besides trypsin and chymotrypsin, it strongly i n ­ hibits plasmin as well as kallikreins o f different origin (Ta­ ble 2) [ 3 - 7 , 2 1 ] . In view o f the therapeutic uses o f aprotinin and its use i n both clinical and experimental animal studies, its species specificity is o f particular interest. Several investigators have found that various tissue kallikreins (from pancreas, sub­ mandibular glands, kidney or urine, and colon) as well as plasma kallikreins from man, pig and cattle are inhibited by aprotinin, whereas the corresponding guinea pig kallikreins are not inhibited [ 5 - 7 ] . Divergent results have repeatedly been obtained w i t h mouse, dog and rat kallikreins [7]. Re­ cently, however, it was shown that urinary kallikreins o f the rat are very effectively inhibited by aprotinin [48]. The therapeutic uses o f aprotinin are based on its i n h i b i t i o n o f human trypsin, plasmin and plasma, tissue or urinary kallikrein. It should be noted that plasmin and tissue k a l l i ­ kreins are inhibited to a decidedly greater extend than the plasma kallikrein (Table 2). The inhibition o f human pancreatic proteinases by aprotinin is o f special interest i n connection w i t h the therapeutic use o f the drug Trasylol i n acute pancreatitis. Human cationic as well as anionic trypsin is strongly inhibited by aprotinin [49, 50]. H u m a n chymotrypsin I I is not inhibited at a l l , while aprotinin appears to have a fairly low affinity for human chymotrypsin I and protease Ε [49]. Protease Ε is quite simi­ lar to porcine pancreatic elastase [51], but not to the " t r u e " (i.e. elastin-digesting) human pancreatic elastase [52]. The affinity o f aprotinin for human pancreatic kallikrein has not been quantitatively estimated. I f the relation between tissue and urine kallikreins in man [44] is as close as that between tissue and urine kallikreins i n the pig [53], aprotinin w o u l d Table 2: Dissociation constants Kj of enzyme-aprotinin complexes. Enzyme Trypsin, bovine Anhydrotrypsin, bovine Trypsinogen, bovine Chymotrypsin, bovine Plasmin, porcine human Kallikrein pancreatic, porcine pancreatic, porcine submand., porcine urinary, porcine plasma, porcine urinary, human plasma, human Elastase, human leukocytes Urokinase, human urine a

b c

Kj (mol/1) 6.0 5 weeks 12 min

7

3

5

b

t* )

7a

8 a

4a

4a

IxIO"

1

8

5

8

13

k-c (s- )

2a

8.5

9

5.4x10" ) 1.4xl0" ) 2 xlO" ) 8

3

11 sec

[36] [75] [78] [79] [36] [36] [77] [79] [74, 79]

') IKL2 in equation (a), normally practically identical with kd. ') 1 Half-life of dissociation. The half-life of association (tljp) is concentration-dependent. For the reaction of excess native aprotinin with trypsin ( I x I O mol/1) ttfyf - 6 . 3 s, and with chymotrypsin ( I x I O mol/1) ti)f ~ 63 s; for the reaction of modified aprotinin (I*) the corresponding values are ίψ ~ 39 min (trypsin) ;and tt)p ~ 9.5 days (chymotrypsin), respectively, at the same enzyme concentration. (Calculated from the values given in Table 3 by F. Fiedler, Munich). -7

-7

A r z z n e i m . - F o r s c h . / Drug Res. 33 ( I ) , Nr. 4 (1983) F r i U z el al. - Aprotinin

483

6

bond approximately 10 times more rapidly than bovine trypsin [79]. Incubation o f aprotinin w i t h 0.2 m o i % o f tryp­ sin 1 at 21 °C at a p H between 6.2 and 7.8 resulted in an equilibrium constant K d close to 1, corresponding to equimolar amounts o f virgin and modified inhibitor i n the incu­ bation mixture. This constant increased exponentially with increasing p H ( K ~ 2.4 at p H 8.5 and K ~ 4.5 at p H 8.9) [79]. A t p H 8.2, the equilibrium was reached after 4.6 days, starting either from virgin or from modified aprotinin. Besides the decidedly higher (approx. 10 times) dissociation rate (see k in Table 3) o f the aprotinin-chymotrypsin com­ plex as compared w i t h the aprotinin-trypsin complex, there are striking differences i n the global association rates for the formation o f the stable complex C from either virgin or mo­ dified aprotinin (Table 3). The reaction o f the modified i n h i ­ bitor w i t h chymotrypsin and trypsin proceeds much more slowly (by a factor o f 10 and 10 , respectively) than with virgin aprotinin (k* = 8.5 and 3 x l 0 MM s-' vs k 6 χ 105 and 3 χ 10 s- , respectively) [77, 78]. This is o f prac­ tical significance i n that the two inhibitors may be quantita­ tively estimated i n mixtures preincubated for different lengths o f time. Whereas, for example, 99.9% o f native aprotinin but less than 0 . 1 % o f modified aprotinin com­ plexes w i t h chymotrypsin, under suitable conditions, w i t h i n 1 m i n , both inhibitor forms are totally complexed w i t h tryp­ sin after 2.5 h o f preincubation [79]. h y

h y d

h y d

4

d

5

2

3

n

5

o n

1

The following observations are also o f general interest: (1) A n y chemical modification o f the ion pair formed between Lys-15 o f aprotinin and Asp-177 o f trypsin i n the specificity pocket results in a significant increase (10 times or more) i n the dissociation rate o f the complex [36]. The marked differ­ ence between the stability o f the trypsin-aprotinin complex and that o f the chymotrypsin complex (see Table 3) is main­ ly due to the additional, stabilizing interactions afforded by the specificity pocket for Lys-15 [36, 77]. (2) The formation of a very tight complex between aprotinin and anhydrotryp­ sin (Table 3) clearly shows that the alcohol side chain o f Ser-183 o f trypsin plays no role i n the stabilization o f the complex [36]. (3) The absolute values o f free energy o f asso­ ciation are very high for the firm complexes, e.g. AG\ = - 1 8 . 1 and - 1 1 k c a l / m o l for the complex w i t h trypsin and chymotrypsin, respectively, whereas the enthalpic values CdH ) are near zero (25 °C, p H 8.0) [36]. 3

e

The p H of the medium has a significant effect on the rate and equilibrium constants shown i n Table 3 and 2 and o n Khyd. N o r m a l l y , the equilibrium constants K j and Khyd as well as the dissociation rate constants kd and k_c are lowest i n the neutral and slightly alkaline p H range ( p H ~ 7 - 9 ) , whereas the association rate constants k and kjn then have the highest values. Accordingly, complex f o r m a t i o n is strongly favored at the optimal p H o f the proteinases. A s the p H o f the medium decreases, K i and kd o r k_c increase sharply, whereas k or k£ decrease significantly so that dissociation of the complex is promoted i n acidic media. The Kj o f the pig pancreas kallikrein-aprotinin complex, for example, increases by a factor o f 10 from p H 7.8 to 5; at p H 4 the complex is completely dissociated [ 4 ] . The K j o f the bovine trypsin-aprotinin complex increases a p p r o x i ­ mately 10 times from p H 8 to p H 3 as estimated from data given i n Refs. [36, 75]. A more specific example is given i n Fig. 3, which clearly shows the marked change i n the asso­ ciation rate constant k and in the dissociation e q u i l i b r i u m constant Kj w i t h increasing p H for the a p r o t i n i n chymotrypsin system [76]. o n

o n

n

3

8

o n

4. Molecular mechanism of inhibition 4.1. Formation and structure of aprotinin-proteinase complexes Our knowledge about the motive forces responsible for the formation of a firm complex o f a proteinase and a proteintype inhibitor is based chiefly on the results o f X - r a y crystal­ lography studies [82-88] as well as N M R (section 6.2.) [89], circular dichroism [90], and kinetic studies [36, 37, 7 5 - 8 0 ] . Complex formation is generally associated w i t h : (1) A per­ fect fit o f the reactive-site inhibitor residue (a lysine or arginine residue in the case of trypsin, plasmin and kallikrein) i n the specificity pocket o f the enzyme; (2) formation o f a tetrahedrally oriented intermediate product by action o f the nucleophilic oxygen of the alcohol group o f the enzymatic active-site serine residue upon the carbonyl group o f the l y ­ sine or arginine residue o f the reactive peptide bond o f the inhibitor; and (3) stabilization o f the adduct by additional reactions i n the vicinity o f the specificity pocket and reac­ tive-site bond residues, respectively. The extended contact area existing between the two poly­ peptide segments of the inhibitor (Posns. 12-18 and 3 4 - 3 9 ) and the various peptide segments o f trypsin (Posns. 3 9 - 4 2 , 57-60, 96-99, 151, 175, 189-195, 2 1 4 - 2 1 6 ; cf. [83, 84]) is shown schematically i n Fig. 4 and, i n greater detail for the aprotinin molecule, i n Fig. 2. Remarkably, 14 out o f 58 amino acid residues i n aprotinin, i.e. 24% o f the total n u m ­ ber of residues i n the molecule, and 24 out o f the 224 resi­ dues o f trypsin (i.e. 10.7%) are tightly packed i n a density comparable to that found i n the interior o f globular protein regions [91]. Consequently, numerous contacts (12 hydrogen bonds, a salt bridge, more than 200 van der Waals inter­ actions, and 8 intermolecular water molecules [83, 84, 92]) contribute to the appreciable gain i n free energy, the driving force for complex formation, so that an extremely high free energy of association of about 18 k c a l / m o l and an associa­ tion constant K ( K = 1/Kj) o f 1.6 χ 10 1/mol result [36]. This high yield o f free energy i n the complexation is possible because the reactive inhibitor domain o f unbound aprotinin already has a structure that almost perfectly complements the contact region at the surface o f trypsin so that only a m i n i m a l adaptation of the two molecules is necessary [87]. This is the principal difference from the reaction of an en­ zyme with a normal substrate chain i n which many degrees of freedom need to be immobilized to permit optimal inter­ action with the catalytic site o f the enzyme. Enzyme-catalyzed peptide bond hydrolysis involves forma­ tion o f an acyl-enzyme intermediate (III) via a covalent tetrahedral adduct (II) before the hydrolysis can be complet­ ed w i t h the aid o f a water molecule (IV) (see Scheme 1 [87, 93]). The reaction is initiated by action o f the nucleophilic oxygen atom of the enzyme's serine residue on the carbonyl carbon atom of the scissile peptide bond (I) as soon as the 13

a

τ

τ

ι

ι

1

1

4

5

6

7

θ

9

1

1

10 pH

Fig. 3: pH dependence of the apparent rate constant of association kon (left ordinate) and of the dissociation equilibrium constant Ki (right ordi­ nate) of the system aprotinin/a-chymotrypsin. Taken from Engel et al. [76]. 484

a

A r z n e i m . - F o r s c h . / Drug Res. 33 ( I ) , Nr. 4 (1983) Fritz e t a l . - Aprotinin

complex is very similar to that o f the trypsin-aprotinin com­ plex [86]. The same is true o f the aprotinin-trypsinogen complex [88]. It appears that enough energy is gained during contact o f the complementary regions to force the disor­ dered, enzymatically inactive conformation o f trypsinogen into the ordered high-energy state o f the active enzyme [90]. Most remarkably, this complex is able to catalyze resynthetis o f modified (reactive-site peptide bond hydrolyzed) aprotinin [94]. The relatively low affinity o f aprotinin for chymotrypsin as compared with trypsin (Table 2) is m a i n l y a reflection o f the poor fit o f the inhibitor Lys-15 residue i n the specificity pocket o f this enzyme [82]. The strong interaction o f this re­ sidue w i t h the specificity pocket o f trypsin, especially be­ tween the Lys-15 a m m o n i u m group and the carboxylate group o f Asp-189 at the base o f the pocket, contributes appreciably to the free energy that is gained during forma­ tion o f the aprotinin-trypsin complex [83, 84, 87]. Figg. 4: Schematic representation of the numerous contacts formed in a proteinnase-aprotinin complex between the amino acid residues of the inhibitor (appprox. 24% of all residues) and the enzyme (approx. 11% of all residues). Thne key area of the inhibitor fits nearly perfectly into the lock area of the enzynme. Painted by A. Hermann according to a space filling α-carbon plot cal­ culated by W. Bode and R. Huber. The X-ray structure of porcine pancreatic kalillikrein and its complex with aprotinin has been elucidated very recently, see- Bode, W., Chen, Z., Adv. Expt. Med. Biol. (Kinins-III. Part A) 156, 289 (19983).

subbstrate is exactly aligned towards the catalytic residues. In thee case o f the aprotinin-enzyme complex, the peptide carbo^nyl carbon (medium-faced, Scheme 1) is only turned in thee direction o f the tetrahedral configuration; i n other wcords, i n the stable complex, an intermediate state between thee initial Michaelis-Menten complex (not shown i n Scheme 1)) and the tetrahedral transition state is frozen, presumably o w i n g to the optimal fit which this conformation offers both prcoteins without any large expenditure o f energy [87]. Cleayagge o f the scissile bond is then hindered by the tight packingg o f the catalytic residues, especially His-57, so that I

traansfer o f the proton from His-57 to H N - A l a - 1 6 of the leavingg group, a necessary step for acyl-enzyme formation (see I I a m d I I I in Scheme 1), is prevented. Although complex for­ m a t i o n is attended by slight distortions in the inhibitor bind­ i n g segments, the reactive-site peptide bond of aprotinin re­ m a i n s intact, therefore. This recently developed concept [87, 921] provides a rational explanation for the low dissociation rattes o f most of the aprotinin-proteinase complexes (see sectioon 3.3.). In view o f the gain in free energy resulting from the numer­ o u s contacts between enzyme and inhibitor it is not surpris­ i n g that, contrary to previous assumptions, complex formatioDn need not always be preceded by formation of a tetrahecdral or acyl-enzyme intermediate. In fact, the enzymaticallly inactive anhydrotrypsin likewise forms a tight complex w i u h aprotinin (Table 2). The structural geometry of this o=c

/

+

\

HN

Enzym ΗΝ — Η

R'

Ν

ΝΗ

4.2. Analogs of aprotinin formed by enzymatic and chemical modification There is now general agreement that aprotinin is a singleheaded inhibitor having the Lys-15 residue i n its active center. The location o f the reactive-site bond w i t h i n the se­ quence is generally demonstrated: (1) By limited proteolysis in a slightly acidic solution, which results i n an equilibrium between the modified (hydrolyzed reactive peptide bond) and virgin inhibitor (reactive peptide bond intact); and (2) by resynthesis o f the virgin inhibitor from the modified i n h i ­ bitor under appropriate conditions [73, 75, 95]. I n the case of aprotinin, however, limited proteolysis o f the Lys-15Ala-16 bond by bovine trypsin could originally be effected only after selective reduction o f the disulfide bridge Cys-14Cys-38 [96, 97]. After reoxidation, the modified inhibitor could be reconverted to virgin aprotinin by means o f trypsin, or-chymotrypsin, plasmin or pancreatic kallikrein, and the localization o f the reactive peptide bond could thus be de­ monstrated also by the conventional route [96]. It has been shown i n the meantime that catalytic amounts o f trypsin, chymotrypsin or plasmin establish a true thermo­ dynamic equilibrium between virgin and modified aprotinin [80]. The modification reaction proceeds very slowly, how­ ever. W i t h plasmin, for example, the equilibrium concentra­ tion between virgin and modified aprotinin is reached, under optimal conditions, after 300 days, starting from either the virgin or the modified inhibitor. In the cases of trypsin and chymotrypsin the reaction is even slower. This also shows, however, that aprotinin essentially reacts like other protein­ ase inhibitor proteins [73]: its reactive-site bond L y s - 1 5 Ala-16 is hydrolyzed by catalytic amounts o f proteinases, and the hydrolyzed bond is subject to thermodynamically controlled resynthesis [80]. Trypsin 1 from the starfish Dermasterias imbricata was re­ cently found to provide easy access to modified aprotinin [74, 79]. I n contrast to starfish trypsin 2 which, i n c o m m o n with bovine trypsin, forms a very long-lived complex w i t h aprotinin, starfish trypsin 1 splits aprotinin rapidly and w i t h high specificity at the reactive-site Lys-15-Ala-16 bond. A l ­ though starfish trypsin 1 also forms a relatively tight com­ plex w i t h aprotinin (Kj - 1 χ 10- mol/1), this complex dis­ sociates much more quickly (t. = 11 s) than the bovine t r y p ­ sin-aprotinin complex (t./ = 10 s). The rates o f reactive pep­ tide bond hydrolysis show a similar difference: starfish t r y p ­ sin 1 splits the Lys-15-Ala-16 bond a m i l l i o n times faster than does bovine trypsin [79]. The modified inhibitor w i t h the hydrolyzed Lys-15-Ala-16 bond was used to replace the Lys-15 residue w i t h other amino acids [96, 98]. As expected, replacement o f Lys-15 by arginine d i d not affect the inhibitory properties o f aprotinin, whereas enzymatic replacement o f this basic residue by phenylalanine or tryptophan produced a significant increase in affinity for chymotrypsin and a decrease i n affinity for trypsin. O f the various aprotinin derivatives (modified also at the y3/y-carboxyl groups o f the aspartic and glutamic acid 9

Τ

/2

7

2

111

His-57 Or=C

+ \

ΗΟ Schheme 1: Enzyme-catalyzed peptide bond (I) hydrolysis involves formation of aan acyl-enzyme intermediate (III) via a covalent tetrahedral adduct (II) De­ force hydrolysis proceeds to completion with the aid of a water molecule (IV). Moodified according to Blow [93] and Huber and Bode [87]. A r z s n e i m . - F o r s c h . / Drug Res. 33 ( I ) , Nr. 4 (1983) Frit;tz et al. - Aprotinin

485

8

Table 4: Reactive site residues of structurally related trypsin-chymotrypsin inhibitors of the aprotinin (Kunitz) type. ++, strong inhibition (Kj ^ IO" mol/l); +, weaker inhibition (Ki > io~ mol/l);-, no inhibition; 0 , not tested (known); ITI, inter-a-trypsin inhibitor. 8

Inhibition of

Position of residues Inhibitor source P Bovine organs: aprotinin Sea anemones, 5 I I Snails, Κ Bovine serum, BI-8 RussePs viper, I I Cobra, Ν NY I I ) Cobra, HHV IF) Cow colostrum Human ITI-14-2 Bovine ITI-14-2 Soybean, Kunitz ) b

d

f) ) ) ) Ρ ) b c

d

r

P

3

13 Pro Pro Pro Pro Arg Leu Leu Pro Pro Pro Ser

2

14 CYS CYS CYS CYS CYS CYS CYS CYS CYS CYS Tyr

P, 15 LysArgLysLysArgLysLys LysArgArgArg-

Ρί 16 Ala Ala Ala Ala Gly Ala Ala Ala Ala Ala lie

p; 17 Arg Arg Ser Ala His Arg Tyr Ala Phe Phe Arg

Kallikrein of p; 18 He Phe Phe Met Leu He He Leu lie He Phe

p;

ρ;

a

3

19 ) He Pro Arg He Arg Arg Arg Leu Gin 0 He

38 CYS CYS CYS CYS CYS CYS CYS CYS CYS CYS

P24

39 Arg Arg Arg Arg Gly Gly Gly Gin Gin Lys

Plasmin Tissue

++ ++ + +

++ ++ ++ ++

e

e

+) 0 e + +

++

)

+) ++ ++

f

r +

>

++

-)

-

Reference

Plasma

+ + 0 + e

+) 0 0 0 0 ++

[17, 18] [102] [103] [12] [104] [42, 105] [42, 105] [106] [Π] [12] [107]

Not involved in the contact with trypsin in the complex. Naja nivea venom. Hemachatus haemachatus venom. Not structurally related. Estimated from titration curves or author's classification (no quantitative data available, see references). Hochstrasser, K., unpublished data (1981).

residues) described recently [99], those w i t h a hydrophobic amino acid residue i n position 15 are interesting inasmuch as they show high affinity for human granulocytic elastase [100]. It is noteworthy that substitution o f Lys-15 by glycine did not entail a significant decrease i n the affinity o f aprotinin for porcine pancreatic kallikrein [100]. The recently accomplished total synthesis o f aprotinin opens the way to a detailed study o f the effect o f amino acid ex­ changes on the inhibitory properties o f this molecule [101]. The results o f studies on the synthesis o f aprotinin, aprotinin analogs, and polypeptide models o f the reactive center o f aprotinin have lately been reviewed [7], 4.3. The structural basis of kallikrein inhibition W h i l e the tertiary structure o f the active site o f the k a l l i kreins remains unknown ), insight into the structural basis for kallikrein inhibition by aprotinin can be gained only by comparison with the inhibitory specificities and reactive-site sequences o f structurally homologous inhibitors (Table 4). Crystallographic studies have revealed that the amino acid residues o f aprotinin that are i n most intimate contact w i t h trypsin i n the complex comprise, i n addition to Cys-14 and Cys-38, the "specificity pocket" residue Lys-15, A l a - 1 6 , and the basic residues Arg-17 and Arg-39 (Fig. 2) [83, 84]. I n view o f the inhibitory specificities of structurally h o m o l o ­ gous inhibitors i t is evident that the relatively strong reac­ tion o f aprotinin w i t h the kallikreins is due to the basic char­ acter o f the residues i n positions 17 and 39 [108]. Replacement o f both basic amino acid residues in these positions by neutral residues completely abolishes the affin­ ity o f an aprotinin-type inhibitor for both tissue and plasma kallikreins (cf. cow colostrum inhibitor). If, however, a basic residue is present i n position 19 (cobra H H V II), k a l l i k r e i n i n h i b i t i n g activity is displayed. It appears, then, that i n an aprotinin-type inhibitor a basic amino acid residue is re­ quired i n one or more o f these positions - 17, 19, 39 - to produce enough energy for complex formation w i t h k a l l i ­ krein. 3

This hypothesis is supported by recent studies o f the struc­ ture/function relation i n an aprotinin-type inhibitor from bovine serum [12] and o f the trypsin-inhibiting a p r o t i n i n type domains (ITI-14-2) derived from human and bovine inter-tf-trypsin inhibitor [ 1 1 , 12] (Table 4). The inhibitor 3

20 ) Arg Arg Gin Arg Arg Ser Ser Arg Leu 0 Ala

Ρ»

) The X-ray structure of porcine pancreatic kallikrein and its com­ plex with aprotinin has been elucidated recently, see: Bode, W., Chen, Z., Adv. Expt. Med. Biol. (Kinins-III, part A) 156, 289 (1983).

486

from bovine serum bearing arginine i n position 39 is a fairly potent inhibitor o f tissue kallikrein, whereas the inhibitory domain ITI-14-2 o f the human inter-a-trypsin inhibitor has no basic residue i n any o f the above-cited positions and so fails to inhibit tissue kallikrein. The I T I - 1 4 - 2 domain de­ rived from the bovine inter-#-trypsin i n h i b i t o r , on the other hand, bears a basic residue i n position 39, lysine, but does not inhibit tissue kallikrein, either (Table 4). I n the latter case, however, a large residue (e.g. tryptophan) i n position 19 may prove a steric hindrance to complex formation. The affinity o f an aprotinin-type i n h i b i t o r for plasmin (or trypsin), as contrasted with kallikrein, is not significantly af­ fected by such replacements o f amino acids at the reactive site. The inhibition of plasma kallikrein and, notably, tissue kallikrein evidently requires additional structural features in specific subsite positions. This is true because o f the more restricted substrate specificity o f the kallikreins as compared with trypsin or plasmin. We may justifiably infer that the active sites o f any o f the kallikreins that form complexes with aprotinin are very similar. A close similarity between porcine and human tissue kallikrein or urinary kallikrein has in fact been reported recently [44]. Yet the decidedly lower affinity between aprotinin and the kallikreins when compared with trypsin (Table 2) indicates that the fit o f the complementary contact regions i n the aprotinin-kallikrein complex is less than perfect, or that the contact area differs markedly from that o f the aprotinin-trypsin complex. This is true at least for contact i n the specificity pocket - replace­ ment of Lys-15 by glycine did not significantly affect the af­ finity o f aprotinin for tissue kallikrein [100]. Kallikrein inhibitors as well as kallikreins are known to oc­ cur in snake venoms [104, 105]. Furthermore, snake toxins may be structurally related to aprotinin without possessing inhibitory activity against known proteinases [109]. The sig­ nificance of these observations is as yet unclear. 5. Special properties relevant to administration of aprotinin 5.1. Interaction with glycoproteins and mucopolysaccharides The relatively high basicity o f aprotinin, which is due to its high isoelectric point o f 10.5 [21], seems to be chiefly re­ sponsible for two special features o f this inhibitor molecule: (1) Its binding to acidic glycoproteins or mucopolysacchar­ ides including heparin [110, 111], and (2) its " f i x a t i o n " and degradation i n the kidney following therapeutic administra­ tion [112-114]. A p r o t i n i n derivatives o f lower basicity [96, 112] and aprotinin-type inhibitors from sea anemones [115] and snails [96, 116] as well as other trypsin inhibitors o f siA r z n e i m . - F o r s c h . / Drug Res. 33 (1), Nr. 4 (1933) Fritz et al. - Aprotinin

m i l i a r molecular weight but lower basicity [112] are excreted in tthe urine. 5.22. Immunogenicity ApDrotinin-specific antibodies have not so far been observed in human sera d u r i n g or after aprotinin treatment [117]. A p ^ r o t i n i n thus appears to be a comparatively weak i m miunogen. Guainea pigs can, however, be sensitized to i t by a special i m m i o n i z a t i o n program [117]. Aprotinin-precipitating antisera hawe also been obtained i n rabbits by immunization w i t h cormplete and incomplete Freund's adjuvant. These antisera wetre identified by immunodiffusion and immunoelectrophcoresis (Eben, A . and Truscheit, E., 1964; data published by Haberland [117]). Other immunization methods for aprrotinin were described recently [10, 13, 14]. A p r o t i n i n speecific antibodies were also obtained when high-molecular weiight cross-linked a p r o t i n i n served as the immunogen [1118]. Ap^rotinin-specific antisera produced according to Eben and Trvuscheit [117] have been used i n immunofluorescence studiess [114] and i n radioimmunoassays [35, 114]. These antiserra were found capable o f neutralizing almost completely the? trypsin-inhibiting activity o f aprotinin when gelatin [1118], casein or BzArgOEt (Truscheit, E., unpublished observation) was used as substrate. 5.3k Elimination after administration Serrum or plasma levels o f aprotinin obtained after i.v. injectiorn i n animals and humans decline rather rapidly owing to disttribution o f the i n h i b i t o r in the extracellular fluid and subsequent accumulation, particularly i n the kidney [7, 1199-121]. The following human serum levels were measureed, for example, per M i l l i l i t e r after i.v. administration o f a siingle bolus o f 500,000 or 50,000 K I U [121]: 0.25 h : 50, 5; (0.5 h : 35, 3.5; 1 h : 25, 2.5; 2 h : 17, 1.7; 4 h : 10, 1. The hallf-life o f e l i m i n a t i o n changed from approximately h/ = 0.7? h 1 h after injection to t> = 7 h 12 h after injection. Aboout 80% o f the a p r o t i n i n dose is found i n the rat kidneys afteer 4 h [119]; a p r o t i n i n is taken up by the epithelial cells o f tthe proximal tubules [113, 120]. It appears that aprotinin is ailmost entirely metabolized in the kidney lysosomes [120] inaäsmuch as biologically active aprotinin is not normally excretted in the urine. Only 1.5% o f 1,000,000 K I U , for example;, was found in human urine a short time (1.5 h) after i.v. injeection [119]. A p r o t i n i n that is covalently bound to soluble- polysaccharides is more slowly eliminated from the circ u l a t i o n and is excreted w i t h the urine without accumulating in tthe renal tissue [122]. Altthough the toxicity o f aprotinin is extremely low - the L D ) for mice and rats is 2.5 χ 10 K I U / k g and that for rabbitss 0.5 χ 10 K I U / k g - rapid i.v. injection o f large doses s h o u l d be avoided, for the high basicity o f aprotinin may cauase liberation o f histamine and lead to an anaphylactoid reatction. 2

/2

6

5 0

6

5.4k Effects on cellular function In w i e w of the widespread use o f aprotinin as a proteinase i n hibiitor, recent observations o f additional biological effects o f thiss inhibitory polypeptide merit special attention. A p r o t i n i m has been found to influence the response of body cells suchh as leukocytes, platelets, and macrophages to various s t i m i u l i as well as the activities o f membrane-associated enzyimes. We know little, however, about the mechanisms o f thesse actions of aprotinin. These effects are briefly discussed in ssection 6.5. 6. EBiochemical and biomedical applications 6.1.. General applications A p i r o t i n i n has proved to be a valuable tool i n biochemical andi biomedical research and i n routine procedures used in thesse areas. It is used extensively for the following purposes: 6.1.. 1. Proteinases inhibited by aprotinin can easily be puri­ fied! by affinity chromatography w i t h insolubilized aprotinin derrivatives [7], e.g. aprotinin-Sepharose [123] and aprotininArznoeim.-Forsch./ Drug Res. 33 ( I ) , Nr. 4 (1983) F r i t z e t al. - Aprotinin

cellulose [15]. Besides trypsin [ 1 2 3 - 1 2 5 ] , chymotrypsin. [123], pancreatic elastase [52], and plasmin or plasmin frag­ ments [126, 127], plasma and tissue kallikreins [7, 44, 1.28, 132], as well as granulocytic elastase and cathepsin G [133, 134] have been successfully isolated by this means under ap­ propriate conditions. Aprotinin-Sepharose has also been used for purification o f a hormone-degrading enzyme [135]. 6.1.2. Aprotinin-sensitive enzymes can be crystallized as aprotinin-proteinase complexes either for purposes o f p u r i f i ­ cation [2] or for X-ray crystallography [ 8 2 - 8 4 ] . For the lat­ ter purpose, complexes o f aprotinin with inactive enzymes such as anhydrotrypsin [86] and trypsinogen [88] have been crystallized as well. 6.1.3. A p r o t i n i n is widely used as a molecular weight marker ( M . W . approx. 6500) i n gel permeation chromato­ graphy [136] and SDS electrophoresis [137] (see e.g. Product Profile 6040SA from Bethesda Research Lab., Rockville, USA). 6.1.4. K a l l i k r e i n activity i n a m i x t u r e o f esterases and p r o ­ teinases can be identified by specific i n h i b i t i o n o f the k a l l i ­ krein w i t h aprotinin. Thus, the presence o f aprotinin (10 K I U / m l , i.e. 2 χ 10- mol/1) in the assay k i t for determina­ tion o f kallikrein activity i n human urine by use o f a syn­ thetic substrate permits a clear distinction to be made be­ tween substrate cleavage induced by kallikrein and by uro­ kinase [138]. 6.1.5. A p r o t i n i n is frequently used to solve a common prob­ lem i n biochemical and biomedical research: how to prevent proteolytic degradation o f proteins or polypeptides, en­ zymes, proenzymes, preproteins, etc. i n biological fluids or tissue extracts and during purification. A p r o t i n i n is used for the following purposes, for example: (a) Suppression o f proenzyme activation during purification o f kininogens [139, 141], and preparation and assay o f plasma samples used for determination o f fibrinopeptide A or Β [142]; (b) prevention o f breakdown o f Proteohormones such as gluca­ gon during the preparation and assay o f samples to be used in radioimmunoassays [ 1 4 3 - 1 4 6 ] ; (c) preservation o f protease-sensitive plasma factors such as clotting factor V I I I (antihemophilic factor) [147] and fibronectin [148] during their isolation, storage and analysis. A p r o t i n i n concentra­ tions used for these purposes ranged from 100 K I U / m l (i.e. approx. 2 χ 1 0 mol/1) to 1600 K I U / m l (i.e. approx. 3 x 1 0 - 5 mol/1); i n most cases 500 K I U / m l (approx. 1 χ 10- mol/1) should be sufficient. 6.1.6. A p r o t i n i n may be used to terminate lysis caused by proteinases i n tissue culture studies (see section 6.3.) and i n experiments i n which only a limited proteolysis is required. Examples are i n h i b i t i o n o f kinin-liberating enzymes i n sam­ ples subjected to biological assays (by 1650 K I U / m l ) [149], and preparation o f defined molecular forms o f plasmin [150, 151]. 7

6

5

6.2. Tool for protein conformation and protein-protein interaction studies A p r o t i n i n , a typical globular protein, has been used i n n u ­ merous studies on the fundamental aspects o f protein con­ formation [22, 152-155]. These have included theoretical and experimental studies o f the internal m o b i l i t y o f proteins, protein folding, and surface interactions w i t h solvents, and they have led to a novel description o f globular protein con­ formation by a dynamic grouping o f rapidly interconverting molecular structures [22, 152, 153]. I t is especially interest­ ing that the average solution conformation o f a p r o t i n i n closely matches the conformation o f its crystalline structure: " H y d r o p h o b i c clusters form stability d o m a i n s " i n the inter­ ior o f the i n h i b i t o r " w h i c h function as pillars for the archi­ tecture o f the protein m o l e c u l e " [153]. I n these studies, furthermore, nuclear magnetic resonance was used w i t h ad­ vantage for the conformational description o f a p r o t i n i n ana­ logs adapted to mechanistic studies o f proteinase-inhibitor interactions [156] and to studies o f the state o f catalytic groups i n proteinases, proenzymes, proteinase inhibitors and their complexes [157, 158]. Extensive knowledge o f the pro487

perties o f aprotinin also permits its application to the solu­ tion o f as yet unsolved problems, such as e.g. a molecular explanation for the dramatic differences i n the dissociation rates o f the complexes o f aprotinin w i t h either bovine tryp­ sin or crayfish trypsin 1 despite the excellent affinity for aprotinin shown by both enzymes (see section 3.3.) [74, 79]. The significance o f aprotinin ( " E . Werle's kallikrein i n activator") as a model compound i n biochemistry and bio­ physics has been further outlined recently by Huber, R., A d v . Expt. Med. Biol. ( K i n i n s - I I I , Part A ) 156, 29 (1983). 6.3. Tissue culture and organ preservation When present in cell culture media i n concentrations rang­ ing from 300 K I U / m l (approx. 6 x 1 0 - 6 m o l / l ) to 10,000 K I U / m l (approx. 2 χ 1 0 m o l / l ) , aprotinin exerts a variety o f effects: (1) I t delays the degradation o f Proteohormones such as sub­ stance Ρ [159] and insulin [160, 161]. Prevention o f insulin degradation by aprotinin accounts for the observed increase i n the absorption o f subcutaneously injected insulin i n the presence o f the proteinase i n h i b i t o r [162]. A p r o t i n i n may thus be successfully used to treat insulin-resistant diabetes [163]. 4

(2) It preserves the integrity and so extends the lifes o f cells and cell membranes from kidney [164] and cerebellum [165]. (3) A dose-dependent i n h i b i t i o n o f D N A , R N A and protein synthesis has been observed i n stimulated mous lymphocytes at aprotinin concentrations between 0.3 χ 10- and 2.5 χ 10m o l / l [166]. (4) I f applied i n relatively small amounts (10 to 500 K I U / m l ) , i t prevents toxic products released from disinte­ grating cells from damaging intact targets [167]. (5) It inhibits the uptake o f adenosine by cardiac cells; this can be important for improved perfusion o f the myocar­ d i u m i n vivo [168]. A p r o t i n i n has also been reported to have an advantageous effect on organ preservation. For kidney preservation, aprotinin (500-2000 K I U / k g body weight) was injected into animals immediately before removal o f the organ, which was then incubated i n an aprotinin-containing medium prior to transplantation [169, 170]. A protective effect o f aprotinin on lung tissue i n vitro has also been described recently [171]. 7

7

6.4. Inhibition of (transformed) cell growth Several groups have investigated the possible usefulness o f aprotinin as an immunotherapeutic agent i n cancer. It was found to inhibit the growth o f malignant transformed cells (at lower doses) but also the growth o f normal cells (at higher doses), aprotinin being bound and endocytosed by the t u m o r targets [172, 173]. A p r o t i n i n administered by various routes depressed the growth and invasiveness o f solid tumors i n animals [174]. Recent studies indicate that this therapeu­ tic effect o f aprotinin may be mediated by improvement i n the host's immune response to tumors [175]. It was noted, for example, that bolus injection o f a high dose o f aprotinin i n both human subjects and animals led to a significant i m ­ provement o f the i n - v i t r o lymphocyte response, and this in t u r n resulted i n reduction o f the tumor-induced lymphocyte suppression [175]. A p r o t i n i n is apparently able to improve cell-mediated i m m u n i t y by direct action on the lymphocyte (see section 6.5.), and the effect is stronger in cancer patients than i n normal subjects. However, not all studies have con­ firmed that aprotinin can i n h i b i t t u m o r growth and invasive­ ness. The conflicting results reported by different authors may be due to a dose and species dependence o f the effect. 6.5. Effects on leukocytes and macrophages A p r o t i n i n affects various enzyme systems o f the leukocytes and the response o f lymphocytes. It inhibits, though rather weakly, a basophil kallikrein-like enzyme that is released (together w i t h histamine) from human peripheral leukocytes upon stimulation w i t h IgE [176, 177]. By means o f aprotinin-Sepharose, leukocytic proteinases generating factors 488

from complement C5 that are chemotactic for leukocytes and tumor cells can be removed from the i n c u b a t i o n m e ­ dium [178]. W i t h the use o f this affinity sorbent, a k a l l i ­ krein-like enzyme was recently isolated from the cytosol o f polymorphonuclear granulocytes [179]. T h i s enzyme seenns to be different from the kininogenase that is believed to be present i n the lysosomal lysate o f the same cells [180]. A p r o t i n i n can either increase (at low doses) or i n h i b i t (at higher doses) the response of peripheral lymphocytes to v a r i ­ ous stimuli but it is toxic to unstimulated cells i n culture [181, 182]. Remarkably, it binds to the plasma membrane o f both peripheral blood lymphocytes and p o l y m o r p h o n u c l e a r leukocytes [181]. Endocytosis o f aprotinin was demonstrable in the latter case. It is therefore conceivable that a p r o t i n i n influences cell function by inhibiting ( i n secondary lysosomes) neutral proteinases that are normally released extracellularly to potentiate lymphocyte stimulation. Leukocytic cathepsin D releases sizable amounts o f l e u k o k i nins, i.e. potent permeability agents, from a precursory p r o ­ tein, leukokininogen, which is found i n ascites f l u i d p r o ­ duced in neoplastic diseases [183]. Cathepsin D can act upon this substrate only after conversion o f a prosubstrate to leukokininogen, a process mediated by a trypsin-like en­ zyme. This proteinase, too, is effectively inhibited b y a p r o t i ­ nin [184]. During macrophage-tumor interaction, aprotinin can i n h i b i t the cytolysis o f the neoplastic targets by activated macro­ phages i f present i n a concentration o f 750 K I U / m l (approx. 1.5 χ 10- mol/l) [185]. The cytolytic activity was found to be generated by a specific proteinase that is secreted by the activated macrophages [186]. 5

6.6. Blood preservation and inhibition of platelet aggregation Platelet aggregation (the second phase, associated w i t h re­ lease o f serotonin) is very effectively inhibited by a p r o t i n i n concentrations between 0.4 χ 10and 1 χ 10mol/l [187-191]. The spontaneous formation o f microaggregates in stored blood is thus prevented, clotting factors are pro­ tected, and fibrinolytic activity is depressed, at least for 5 days. Under transfusion conditions, platelet function is immediately restored so that the patient's coagulation status remains unchanged [187, 189]. Furthermore, a bolus injec­ tion o f 20,000 K I U / k g body weight o f aprotinin can lessen the tendency to aggregation ordinarily observed after major surgery [192]. 5

5

6.7. Wound healing It has been reported that aprotinin reduces the development of adhesions and o f secondary necrosis after operations [193, 194]. The inhibitor is also used as an additive to adhesive fibrin ("fibrin glue") for adapting tissues and sealing bleed­ ing areas with fibrin, preventing its dissolution before tissue repair has set i n [195-197]. 6.8. Tool for studies of muscle metabolism and renal function Glucose uptake by working human muscle cells is signif­ icantly reduced after aprotinin administration (bolus injec­ tion o f 500,000 K I U / 1 0 m i n ) but returns to n o r m a l upon concomitant administration o f bradykinin (13 ng/min) [198-200]. High doses o f aprotinin (125,000 K I U / k g i.v.) also delayed appreciably the metabolic rehabilitation o f rat skeletal muscle during recirculation following ischemia [201]. A rational explanation for these observations is the i n ­ volvement o f kallikrein or a kallikrein-like enzyme i n the mediation o f insulin action on glucose uptake by skeletal muscle by way o f kinin liberation [200]. The administration o f aprotinin to volume-expanded rats leads to a significant reduction o f glomerular filtration rate, clearance, urine volume, and urinary immunoreactive P G E [202]. This finding suggests that the kallikrein-kinin system may contribute to changes i n renal function during extracel­ lular volume expansion. A p r o t i n i n is therefore a valuable tool in studies on the involvement o f the kallikrein-kinin 2

A r z n e i m . - F o r s c h . / Drug Res. 33 ( I ) , Nr. 4 (1*83) Fritz et a l . - Aprotinin

sysstem i n the body's regulatory mechanisms. Besides kallikrceins a n d kininogenases, however, other enzyme systems maay be affected by aprotinin. A n example is the human kid­ n e y aminiopeptidase o f the brush border membrane. This enzyrme is activated by aprotinin i n its membrane-bound form b u i t inhabited by i t after solubilization [203]. 6.S9. Effects on fertilization Proteases p l a y a significant role i n the fertilization process [2(04, 2 0 5 ] . T h e trypsin-like acrosomal proteinase acrosin, forr e x a m p l e , assists the spermatozoon in penetrating the zoma p e l l u c i d a o f the o v u m ; trophoblast and uterine proteinasees a i d i n i m p l a n t a t i o n o f the blastocyst i n the uterus. Pro­ teinase i n h i b i t o r s could therefore be utilized to prevent con­ ception.. Recent investigations have shown that in-vivo use of; a p r o t i n i n for the blockade o f acrosin is difficult because o f thee high concentration (approx. 1 χ 10- m o l / l ) that is needed i f t t h e i n h i b i t o r is to penetrate the sperm head membranes [644]. I n rabbits, however, implantation could by prevented w i i t h miuch lower concentrations o f aprotinin when this was inttroduced into the cavum uteri [206]. This contraceptive effecct o f a p r o t i n i n may result from inhibition o f a trypsin-like or k a l l i k r e i n - l i k e proteinase o f the rabbit trophoblast that plaays a role i n the initiation o f embryo implantation i n the uteerus [ 6 5 ] . Inifusion o f aprotinin i n pregnant rats led to a reduction o f uteerine m o t i l i t y and a significant prolongation o f parturition b y c o m p a r i s o n w i t h saline-infused controls [207]. A p r o t i n i n is o f benefit i n cases o f menorrhagia and intramenstrual spootting due to the use o f intrauterine devices [208]. Repeat­ ed! injections into the uterine cavity stop the bleeding and maarkedly reduce inflammatory reactions including pain. Prrior intravaginal administration o f aprotinin has been repo)rted t o preserve the integrity o f cells i n vaginal smears [2C09]. 2

t i o n o f the |

Fibrinolysis

t

HMW .' Kininogen ;

|

Η Complement system]

Scheme 2: Solid-phase activation of the intrinsic coagulation pathway: in­ teractions between clotting, fibrinolysis, complement, and the kin in-gener­ ating system [242, 243]. Plasma kallikrein accelerates activation (+, positive feedback), whereas the histidine-rich peptide, cleaved off from high-molecular weight (HMW) kinin­ ogen by plasma kallikrein, inhibits activation (-, negative feedback). HMW kininogen binds factor X I and plasma prokaliikrein (both occur in plasma associated with it) via the histidine-rich peptide to the negatively charged sur­ face, an exposed collagen fiber or basal membrane, lipopolysaccharides, glass, kaolin, etc. Aprotinin can inhibit the action of both plasmin and plama kalli­ krein, as indicated by circles in the pathways.

anism o f action. A p r o t i n i n is readily available i n p u r e form and is therefore widely used in various research d i s c i ­ plines and routine assays. It has also become a v a l u a b l e d r u g for the treatment o f a variety o f diseases. The i n f o r m a t i o n about functional aspects o f aprotinin remains scanty, h o w ­ ever. The following aspects are o f special interest for future re­ search: (1) The molecular basis for the pharmacologic effects of aprotinin not associated w i t h proteinase i n h i b i t i o n , i n ­ cluding its interactions w i t h membranes; (2) e v a l u a t i o n o f optimal dosage for interfering either w i t h the c o a g u l a t i o n / kinin-liberating or the fibrinolytic pathway i n v i v o ; (.3) eval­ uation o f optimal dosage for inhibition o f tissue k a l l i k r e i n s and/or other proteinases such as trypsins and c h y m o t r y p s i n s liberated during local inflammatory processes; (4) the func­ tional significance o f the occurrence o f a p r o t i n i n i n the h i g h ­ ly specialized tissue mast cells throughout the b o v i n e organ­ ism; and (5) the functional significance o f the a p r o t i n i n - l i k e inhibitor that is present i n bovine serum and o f the aprotinin-type polypeptide domains i n the inter-a-trypsin i n h i b i ­ tor. Elucidation o f the last two questions is likely t o open additional avenues o f aprotinin research and future m e d i c a l applications. 9. Appendix on aprotinin applications

H o w these enzymes interact w i t h other coagulation factors or fibrinolytic factors is shown diagrammatically i n Scheme 2 [242, 243]. Since aprotinin has a significantly greater affinity for plas­ m i n (see Table 2), i t should be possible to regulate the de­ sired effect, viz. i n h i b i t i o n o f either fibrinolysis or blood clotting, by commensurate dosing o f the inhibitor [244]. This presupposes knowledge o f the actual concentrations o f all the reactants i n relation to time, under in-vivo condi­ tions, and these concentrations cannot at present be deter­ mined because o f the lack o f appropriate methods. They can, however, be calculated for systems containing only plasminogen, plasma prokaliikrein, and aprotinin [245]. In this case aprotinin w i l l be bound to liberated plasmin up to equimolar concentrations inasmuch as the dissociation con­ stant o f this complex is far below the in-vivo plasmin/plasminogen concentration. The in-vivo concentration o f plas­ ma prokaliikrein, however, approaches the Kj-value o f the aprotinin-kallikrein complex. Therefore, a large molar ex­ cess o f aprotinin is needed to obtain complete inhibition o f liberated plasma kallikrein. This can be simulated i n a test system measuring contact activation o f the coagulation cas­ cade, which is stimulated by plasma kallikrein. Strong i n h i ­ b i t i o n is obtained only at aprotinin concentrations ranging from 250 to 500 K I U / m l , equivalent to a five to tenfold mo­ lar excess o f aprotinin over plasma prokaliikrein resp. kal­ likrein [245, 246]. For complete inhibition o f plasmin i n the pure system, an aprotinin concentration o f 125 K I U / m l is sufficient provided all o f the plasminogen is transformed into plasmin [245]. Smaller quantities o f aprotinin should suffice under in-vivo conditions since small amounts only o f the proenzymes are activated ordinarily and most o f the lib­ erated proteinases are rapidly inhibited and eliminated by the natural inhibitors, which are present i n molar excess over the proenzymes/enzymes [247]. It is noteworthy that aprotinin also effectively inhibits - i n addition to plasmin - the plasmin-streptokinase complex w h i c h is an intermediate i n plasminogen activation during thrombolytic therapy w i t h streptokinase [41]. Even though the i n h i b i t i o n o f this complex proceeds comparatively slow­ ly (at 1 μπιοΙ/\ aprotinin, the association half-life is 250 s; ki = 1.1 χ 10« 1 m o H s- and k = 1.1 χ 10 s- ; cf. section 3.3.), aprotinin may be used to control bleeding complica­ tions following thrombolytic therapy w i t h streptokinase [41].

9.1. General comments 9.1.1. Availability Aprotinin is available as a sterile solution in ampoules (Trasyiol®) from Bayer and from FBA-Pharmaceuticals. For research purposes it may also be obtained in lyophilized form. It is identical with BPTI (basic pancreatic trypsin inhibitor), PTI (pancreatic trypsin inhibi­ tor (Kunitz)), kallikrein inhibitor from bovine organs, etc. [21]. Aprotinin preparations may contain. ATPase inhibitors [248] but the Trasyiol commercially available has been free of this agent for several years now (A. Arens, personal communication, 1980). 9.1.2. Storage Aprotinin may be stored in lyophilized form below 4 °C for an "un­ limited" time without losing its inhibitory activity. Aprotinin solu­ tions in salt-buffer media, pH 5-8, are stable at least for one month at 4 °C and below -20 °C for years; the same holds true for open am­ poules. During storage in solution, bacterial growth should be pre­ vented by addition of preservatives such as sodium azide, benzyl al­ cohol, pentachlorophenol, thiomersal, and antibiotics. However, a possible direct pharmacologic effect of the preservative has to be taken into account when biological studies are performed. For biochemical assays preservatives need not be added. Aprotinin solutions of pH < 4 and > 9 should be used within a few hours. Direct exposure of aprotinin solutions to sunlight, UV-light and reducing agents should be avoided. 9.1.3. Dialysis, ultrafiltration and chromatography Aprotinin penetrates readily ordinary dialysis tubes but not acetylated ones. In the absence of a suitable salt concentration (below ap­ prox. 0.1 mol/1 NaCl) it may be bound to negatively charged sur­ faces of cell membranes and solid supports, thus giving rise to unspecific effects or loss of aprotinin (e.g. in gel permeation chroma­ tography or ultrafiltration). 9.1.4. Interaction with heparin Under in-vitro conditions heparin may associate with aprotinin, causing turbidity or precipitation. This can be avoided by increasing the salt concentration of the solution. As a precaution, aprotinin should not be used in chromogenic substrate assays in the presence of heparin, or mixed with heparin for blood sample collection and infusion solutions. Under in-vivo conditions such an interference between aprotinin and heparin is not to be expected because of :he low aprotinin concentration in circulating blood. On the other hand, the combined use of aprotinin and heparin may have a beneficial therapeutic effect [249].

8. Synopsis

9.1.5. Immunoassays In immunodiffusion experiments the antibody should be applied to the gel about 4 h before aprotinin because of the rapid diffusion rate of the latter. Possible adsorption of aprotinin on agarose loaded with negatively charged groups should be borne in mind.

The data and observations we have cited show that we al­ ready have detailed knowledge o f the chemical, physical and biochemical properties o f aprotinin and its inhibitory mech­

9.1.6. Inhibition studies In inhibition assays preincubation of aprotinin with the enzyme for 5 min (with kallikrein for 30 min) at the optimum pH of the pro-

1

3

1

2

490

A r z n e i m . - F o r s c h . / Drug Res. 33 ( I ) , Nr. 4 (1*83) Fritz et al. - A p r o t n i n

teirinase is generally sufficient to achieve complex formation. As a Ionnger incubation does not normally affect aprotinin because of its stahbility against proteolytic degradation, the consistency of the degre 10) and in acicidic solution (pH < 5, chymotrypsin and kallikrein; pH < 3, tryf/psin and plasmin). 9.11.7. Use in biological studies At t present it is not feasible to recommend precise aprotinin concentratitions sufficient for totally inhibiting proteinases in biological fluiiids and tissue extracts. The optimum aprotinin amount needed muust be determined individually in each case. However, 1 χ 10 mool/1 (approx. 500 KIU/ml) of aprotinin should be sufficient in moost cases. Approtinin solutions should be tested for their inhibitory activity befonre use. A simple trypsin inhibition assay with the substrates BzMrgpNA (L-BAPA) [27, 28] or BzArgOEt [33] (possibly in com­ bination with alcohol dehydrogenase and N A D [250]) is suitable for thiss purpose. -5

9.22. Inhibition assays 9.22.1. Photometric assay Α ι modification of the originally described trypsin DL-BAPA test [211, 27] has been reported. In this assay aprotinin inhibits the trypsin-i-catalyzed hydrolysis of Ntf-benzoyl-L-arginine-p-nitroanilide (BzzArgpNA or L-BAPA), which is followed photometrically at 4055 nm. One trypsin unit (UBAPA) corresponds to the hydrolysis of 1 juumol substrate per min, i.e. a Δ A^s/min of 3.32 for a 3 ml volume at aa 1 cm light path. One inhibitor unit (IUBAPA) decreases the activ­ ity / of two trypsin units by 50%, which corresponds arithmetically to the ι inhibition of 1 UBAPA of trypsin. The specific inhibitor activity of aaprotinin is given in lUßAPA/mg polypeptide. Reaagents Subbstrate: 50 mg L-BAPA · HCl in 100 ml distilled water Bufiffer: 0.2 mol/1 triethanolamine · HCl/NaOH, 0.02 mol/1 CaCl , pH 7.8 Tryypsin: 10 mg hog trypsin ( ^ 2 UßAPA/mg) or 18 mg bovine trypsin ( ^ 1.2 UßAPA/mg) dissolved in 100 ml 0.0025 η HCl, stored at 4 C Inhhibitor: 10-15 mg aprotinin dissolved in 1000 ml buffer or ap­ prox. 80 KIU/ml (in the case of bovine trypsin approx. 130 KIU/ml) 2

e

Proocedure Plaace thermostated (25 °C) 3-ml cuvettes in a (spectro) photometer. Pippet in the following order: 0.1 ml trypsin solution, 1.8 ml buffer, 0.1 ml inhibitor solution, each prewarmed to 25 °C; mix with a plas­ tic \ spatula and pre-incubate the mixture for 3 min. Start L-BAPA hyddrolysis by adding 1 ml of substrate solution, mix well and record the: linear (!) increase in absorbance at 405 nm for at least 5 min. Reppeat or make a parallel determination with buffer instead of inhibitoor solution ("reference sample"). Calculation 1 mnUßAPA of trypsin corresponds to a A A of 0.00332/min (reference sarrmple). Reduction of the trypsin activity by 50% corresponds to 0.5 mllUßAPA aprotinin. Calculate the specific activity in IUßAPA/mg inhibitor. Biological units (KIU) of aprotinin may be calculated from the Ϊ IUBAPA by the following equation: IUBAPA χ 615 = K I U .

If boovine trypsin ( ^ 1.2 UßAPA/mg) is used in the assay instead of hogg trypsin, K I U are calculated by: IUBAPA Χ 1025 = KIU. 9.2.!.2. Titrimetric assays Thee proposed method is a modification of the test described pre­ viously [25, 26]. In this assay, aprotinin inhibits the trypsincataalyzed hydrolysis of Να-benzoyl-L-arginine ethyl ester (BzArgOE£t, BAEE), and this is followed by titration of the liberated carboxyyl groups (BzArgOH) with an automatic titrator or manually. Onee F.l.P. unit (UFIP) of trypsin corresponds to the hydrolysis of 1 μιπιοί of substrate per min under the given conditions. One UFIP of aprcotinin is defined as the quantity of inhibitor which inhibits two UFIIIP of trypsin by 50%. 1 UFIP of aprotinin corresponds to 30 KIU aprcotinin, I mg pure aprotinin to approximately 238 UFIP or 7143 KIIU. Reaagents Borrate buffer: Dissolve 572.2 mg of disodium tetraborate-10hyddrate and 2.94 g of calcium chloride-2-hydrate in approx. 900 ml distt. water. Adjust the pH to 8.00 (25 C) with 0.1 Ν HCl, fill up withh dist. water to a 1000 ml final volume, and check the pH again. e

Arznneim.-Forsch./ Drug Res. 33 ( I ) , Nr. 4 (1983) Fritz ζ et al. - Aprotinin

Trypsin solution: Dissolve approx. 20 mg trypsin (from pigs with a minimal activity of 45 Unp/mg) in 10 ml 0.001 Ν HCl, store at 0-4 °C. This solution must be prepared daily or kept frozen until used. Substrate: Dissolve 68.56 mg BAEE · HCl in 10 ml dist. water, store at 0-4 °C. This solution must be prepared daily or kept frozen until used. Equipment pH Meter with an accuracy of 0.02 pH units; glass and calomel elec­ trodes; closable titration vessel with temperature-controlled jacket; magnetic stirrer; temperature-controlled heating unit for the reac­ tion vessels (25 ± 0.1 °C); 0.5 ml microburettes; in addition, for automatic titration a control unit for pH state titration, a 0.25 ml motor burette, and a recorder. Complete titration units are supplied by several companies, e.g. Radiometer, Copenhagen, and Metrohm, Herisau. Procedure Preincubation samples Use an aprotinin solution containing approx. 1000 KIU/ml. Prepare the following mixtures in 5 ml tubes: (a) 3.9 ml borate buffer, 0.1 ml trypsin solution (reference sample); (b) 3.5 ml borate buffer, 0.2 ml trypsin solution, 0.3 ml aprotinin solution (inhibitor test sample). Close the tubes, shake and incubate for 10 min at 25 °C. Automatic titration: Fill 1.8 ml of borate buffer and 0.2 ml of sub­ strate solution into the titration vessel and stir for 5 min to maintain constant temperature. Adjust the pH to 8.00 by addition of 0.02 Ν NaOH and add quickly 0.2 ml of either sample (a) or (b). Maintain the pH between 7.95 and 8.05 by continuous addition of 0.02 Ν NaOH. Record the NaOH consumption for at least 6 min. Manual titration: Fill 9.0 ml of borate buffer and 1.0 ml of substrate solution into the titration vessel and stir as for 5 min to maintain constant temperature. Adjust the pH to 8.00 by addition of 0.1 Ν NaOH and add quickly 1.0 ml of either sample (a) or (b); at the same time start a stopwatch. Maintain the pH between 7.95 and 8.05 by continuous addition of 0.1 Ν NaOH. Record the NaOH consumption for at least 6 min. Repeat each test at least once. Calculation of units Determine the NaOH consumption per min graphically from the titration curve (aut, automatic titration; man, manual titration): U (μηηοΐ/min) =

or

U = μπιοί NaOH consumed per min; V = volume of NaOH consumed during test; t = test time in min. Calculate the biological units by the formula: (2 U - U ) x 4 χ F x 30 KIU/ml = 0.3 xc r e f

i n h

Uref = Uinh = F = c =

activity of reference sample; activity of inhibitor test sample; dilution factor; volume in ml of pre-incubation sample (a) or (b), cf. Proce­ dure. For each U f and U i n h , mean values of 4 determinations should be used. The numerical proportion of the values U f and Uinh should be be­ tween 0.8 and 1.2. r e

r e

10. References [1] Kraut, H , Frey, Ε. Κ., Werle, Ε., Hoppe-Seyler's Ζ. Physiol. Chem. 192, 1 (1930) - [2] Kunitz, Μ., Northrop, J. Η., J. gen. Phy­ siol. 19, 991 (1936) - [3] Frey, Ε. Κ., Kraut, Η., Werle, Ε., eds., Kallikrein-Padutin, pp. 157-168. F. Enke Verlag, Stuttgart (1950) - [4] Frey, Ε. K., Kraut, H , Werle, E., eds., Das Kallikrein-Kinin-System und seine Inhibitoren, pp. 114-142. F. Enke-Verlag, Stuttgart (1968) - [5] Vogel, R., Trautschold, I . , Werle, E., eds., Natural Pro­ teinase Inhibitors, pp. 76-95. Academic Press, New York (1968) [6] Vogel, R., Werle, E., in: Bradykinin, Kallidin and Kallikrein Handb. Exp. Pharm., Vol. 25, Erdös, E., Wilde, A. F., eds., pp. 213-249. Springer-Verlag, Berlin (1970) - [7] Vogel, R., in: Bradykinin, Kallidin and Kallikrein - Handb. Exp. Pharm., Vol. 25 Suppl., Erdös, E. G., ed., pp. 163-225, Springer-Verlag, Berlin (1979) - [8] Chauvet, J., Acher, R., FEBS Lett. 23, 317 (1972) - [9] Wilusz, T., Wilimowska-Peilic, Α., Mejbaum-Katzenellenbogen, W., Acta Biochim. Polon. 20, 25 (1973) - [10] Rifkin, D. B., Crowe, 491

R. Μ., Hoppe-Seyler's Ζ. Physiol. Chem. 358, 1525 (1977) - [11] Wächter, Ε., Hochstrasser, Κ., Hoppe-Seyler's Ζ. Physiol. Chem. 360, 1505 (1979) - [12] Hochstrasser, K., Wächter, Ε., FEBS Lett. 119, 58 (1980) - [13] Fritz, H., Krück, Η., Russe, J., Liebich, Η. G., Hoppe-Seyler's Ζ. Physiol. Chem. 360, 437 (1979) - [14] Shikimi, T., Kobayashi, T., J. Pharm. Dyn. 3, 400 (1980) - [15] Fritz, H., Brey, Β., Müller, Μ., Gebhardt, Μ., in: Proc. Int. Res. Conf. on Pro­ teinase Inhibitors, Munich, Nov. 1970, Fritz, H., Tschesche, H., eds., pp. 28-37, W. de Gruyter, Berlin (1971) - [16] Schultz, F., Naturwiss. 13, 338 (1967) - [17] Kassell, B., Laskowski, M . , Biochem. Biophys. Res. Commun. 20, 463 (1965) - [18] Anderer, F. Α., Hörnle, S., J. Biol. Chem. 241, 1568 (1966)- [19] Huber, R., Kukla, D., Rühlmann, Α., Steigemann, W., in: Proc. Int. Res. 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