Isolation of Proteinases and Proteinase Inhibitors by Affinity ...

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Tesser, G. I., Fisch, H. U. & Schwyzer, R. (1972) FEBS Lett. 23, 56-58. Isolation of Proteinases and Proteinase Inhibitors by Affinity. Chromatography on Cellulose ...



Fig. 2. Biologically active ACTH derivative covalently bound to a preparation of polyacrylamide beads

(average value of three independent preparations). The concentration of the hormone derivative in the packed column (phosphate buffer, pH7.5) was 96pmol/litre. Tesser, G. I., Fisch, H. U. & Schwyzer, R. (1972) FEBS Lett. 23, 56-58

Isolation of Proteinases and Proteinase Inhibitors by Affinity Chromatography on Cellulose Matrices GERT WUNDERER,* HANS FRITZ,* WOLFGANG BRUMMER,? NORBERT HENNRICHt and HANS-DIETER ORTHt * Institut fur Klinische Chemie und Klinische Biochemie der Universitit Miinchen, 0 - 8 Miinchen 2 , Federal Republic of Germany and t Biochemische Forschung E. Merck, 0-61 Darmstadt 2, Federal Republic of Germany Purification of proteinases and proteinase inhibitors by affinity chromatography is based on selective adsorption from crude tissue extracts on to matrix-linked derivatives 1974



of the respective antagonists. Contaminating material is removed by washing with buffered salt solutions, and dissociation of the insoluble complex is achieved either under acidic conditions or by treatment with denaturants or synthetic competitive inhibitors (Fritz et al., 1971). We find CM-cellulose (E. Merck, Darmstadt, Germany) a very suitable support for affinity chromatography in batch and in column procedures because it combines chemical and mechanical stability with high capacity. For the adsorption of proteins on matrix-bound protein effectors no spacers are needed. Low-molecular-weight effectors were linked to a new spacered cellulose as described below. Purification of protein proteinase inhibitors from sea anemones, seminal plasma and spermatozoa was achieved by affinity chromatography on immobilized trypsin (E. Merck), bound through its amino groups to carboxyl groups (amide bond) of CMcellulose. Very diluted and contaminated trypsin-inhibitor solutions, obtained from alcoholic extracts of sea anemones, could be enriched more than 100-fold to almost pure isoinhibitor mixtures, by batchwise adsorption on such immobilized trypsin derivatives followed by washing and subsequent elution with acidic salt buffer solutions (see Table 1). Throughout the 16 experiments the same 8g of trypsin-CM-cellulose were used repeatedly. From 11 litres of crude extracts about 250mg of trypsin inhibitors could be adsorbed and eluted quantitatively (Fritz et al., 1972a). Comparable results were obtained by atfinity chromatography of two trypsin inhibitors prepurified from human spermatozoa. In 9 experiments with columns of trypsin-CMcellulose, 40-fold purification and 83-100% yield was achieved (Schiessler et al., 1974). Similar results were observed during the isolation of trypsin inhibitors from seminal plasma. The yields of acid-stable seminal plasma inhibitors could be improved by prior separation of acid-labile inhibitors before batchwise adsorption on to trypsin CMcellulose (H. Fritz & M. Meier, unpublished work). An index of the suitability of affinity chromatography as a mild isolation procedure for labile proteins is the purification of serum and organ kallikreins and of acrosin. A 100-350-fold purification of serum kallikreins is achieved by adsorption on columns of Kunitz soya-bean trypsin inhibitor immobilized on CM-cellulosefollowed by desorption with either urea or benzamidine solutions. The yield of about 70% includes a further gel filtration step (Fritz et al., 1972b).

Table 1 . Batchwise isolation ofprofeinase inhibitorsfrom sea anemoneson CM-cellulosetrypsin Humid trypsin-CM-cellulose (8g) was suspended in the given volume (given in litres in parentheses) of extract of sea anemones containing the inhibitory activity shown in the Table. (One inhibition unit inhibits the activity of one unit of trypsin with Nu-benzoylL-arginine p-nitroanilide as substrate.) The inhibitor-trypsin-cellulose complex was separated by centrifugation and washed with salt-buffer solutions, pH7.8. Afterwards the inhibitor was dissociated from the complex by repeated extraction with acidic salt solutions, pH1.8. Further experimental details are given by Fritz et uf. (19720). Inhibitory activity c

Experiment no. 1 2 3,495 6 7 8 9-16 Vol. 2

Crude extract (inhibition units) 765 (3) 1030 (4) 1500 (8) 1226 (10) 768 (11) 545 (6) 195-857 (1.5-9)


Adsorbed Eluted (inhibition units) (%) 765 64 1000 84 1450 80,82,85 1146 100 768 100 545 100 187-857 91-100

Number of extractions at pH1.8 5

6 5,697 4

4 4 3-8



6 7, 8 , 9 10, 11, 12 13 14

Expt. no.

Pancreas Pancreas Submandibular gland Pancreas Urine Urine Trypsin (bovine)

Kallikrein from porcine

5.0 5.0

1.3 1.3 8.3



206 159 160 145 106

Specific activity , [units/(mlx EZs0)] (units)

104 53 53 75


119 106




94 80 87 79


48 42 23




bits/(ml x E2d1


77 78, 87,97 86, 87, 92

Specific activity Eluted

125 98,95,95 71, 73,58

Bound (units)

The inhibitor-CM-cellulose complex was suspended in various kallikrein solutions, pH7.8, of known activity (see under 'Employed' in the Table). The kallikrein-inhibitor-CM-cellulose complex was separated by centrifugation and thoroughly washed with salt-buffer solutions, pH7.8. Subsequently, kallikrein was dissociated from the complex by repeated extraction with salt solutions, pH7.7, containing 0.5~-beruarnidinehydrochloride. The total kallikrein activity eluted in each of the successive experiments is given as a percentage of the activity bound to the inhibitorcellulose. The specific activities of the kallikreins determined before and after the affinity step are also presented. Further experirneatal details are given by Fritz & Forg-Brey (1972).

Table 2. Batchwise binditg of organ kallikreins to water-insoluble guanidinated basic pancreatic trypsin inhibitor-CM-cellulose and dissociation with 0.5wbenzamidine sokrtion, pH7.8



2 ? 2

8 E 2.e

ij > r







Organ kallikreins were adsorbed by guanidinated basic pancreatic trypsin inhibitor linked to CMcellulose. (The active centre, lysine-15, of basic pancreatic trypsin inhibitor was converted into a guanidino group, otherwise it would be fixed on CMcellulose and the inhibitor thus inactivated.) The data for the experiments with various organ kallikreins are given in Table 2 (Fritz & Forg-Brey, 1972). For the isolation of acrosin, Caminobenzamidine bound to a new cellulose matrix was used (Orth et al., 1974). Highly substituted water-soluble CM-cellulose, after conversion into the corresponding acid azide, is treated with an excess of aliphatic a,o-diamines, resulting in partial cross-linking and in substitution with aminoalkyl side groups. The resulting porous water-insoluble aminoalkyl cellulose matrix is succinylated. 4Amino benzamidine can then be linked by using water-soluble carbodi-hide. This method proved to be superior with respect to capacity and porosity of the matrix. Sperm acrosomal proteinase (acrosin) was isolated from boar spermatozoa by extraction of washed ejaculated spermatozoa with 2 % acetic acid, followed by separation of acrosin from acrosin inhibitors by gel filtration in acidic solution and affinity chromatography of acrosin on the benzamidine-succinyl-aminoalkyl-cellulosecolumn. The adsorption of acrosin on immobilized benzamidine was achieved at pH7.8 and an ionic strength of 1.1. Desorption was achieved with acidic solutions of pH2.0-3.0. This resulted in a 7-15-fold purification with a yield of about 80% (Schleuning et al., 1973). Fritz, H. & Forg-Brey, B. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 901-905 Fritz, H., Forg-Brey. B., Miiller, M. & Gebhardt, M. (1971) Proc. lnt. Res. Conf Proteinase lnhibitors (Fritz, H. & Tschesche, H., eds.), pp. 28-37, W. de Gruyter, Berlin and New York Fritz, H., Forg-Brey, B. & Eress, L. (1972a) Hoppe-Seyler'sZ. Physiol. Chem. 353,19-30 Fritz, H., Wunderer, G. & Dittmann, B. (19726) Hoppe-Seyler'sZ. Physiol. Chern. 353,893-900 Orth, H. D., Briimmer, W. & Klockow, M. (1974) Z. Klin. Chem. Klin. Biochern. 12,241 Schiessler, H., Arnhold, M. &Fritz, H. (1974) in Bayer Symp. V: Proteinaselnhibitors (Fritz, H., Tschesche, H., Greene, L. J. & Truscheit, E., eds.), Springer, Berlin, Heidelberg and New York, in the press Schleuning, W.-D., Schiessler, H. & Fritz, H. (1973) Hoppe-Seyler's Z . Physiol. Chem. 354, 550-554

Biospecific Affinity Chromatography of Sweet-Potato P-Amylase PER VRETBLAD Research Department, Pharmacia Fine Chemicals AB, Box 175, S-751 04 Uppsala 1, Sweden

There are relatively few methods available for the immobilization of biospecificaffinity chromatography ligands which contain no functional groups other than hydroxyl groups. However, this can be achieved by reaction of the ligand at high pH with a polysaccharide matrix which has been previously activated by a bifunctional epoxide. Such activated matrices also bind amino groups, and the immobilization of peptides and proteins by using the same procedure at weakly alkaline pH has been demonstrated (Sundberg & Porath, 1974).The conjugates obtained by this method have several attractive properties: the matrix backbone is practically uncharged, and no charges are introduced by the coupling reaction. Further, the ligand is connected by a strong ether linkage to a spacerarm of essentially hydrophilic nature. Sepharose 6B (4g), activated by 1,4-bis-(2,3epoxypropoxy)butane and stored in a freeze-dried state, was swelled and reacted with 300mg of cyclohexa-amylose (a 8amylase inhibitor) in 12mI of 0.1 M-NaOH at 45°C overnight. The gel was washed and equilibrated with O.OSM-acetate buffer, pH4.8. A mixture of sweet-potato j?-amylase (1.5mg) and bovine serum albumin (3mg) in 2ml of buffer was passed through a 5ml column of the adsorbent. The enzyme was selectively adsorbed and could be displaced by VOl. 2

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