conditions described above, (b) Keratin (20g) was added to 100mlof distilled water in a flask, and the suspension was vigorously stirred for 1 hr. The suspension ...
Agric.
Biol.
Chem.,
55 (9),
2251-2258,
1991
2251
Purification and Properties of a Novel Surface-active Agent- and Alkaline-resistant Protease from Bacillus sp. Y Hisao Shimogaki,*1 Keiji Takeuchi,* Takashi Nishino, Motoyasu Ohdera,* Toshihiro Kudo,* Kenkichi Ohba,* Masanori Iwama** and Masachika Irie** * Biological Science Laboratories, Lion Corporation, Tajima 100, Odawara, Kanagawa 256, Japan
**Department of Microbiology,
Hoshi College of Pharmacy, Ebara 2-4-41,
Shinagawa-ku,
Received
February
Tokyo 142, Japan
12, 1991
In the course of a search for an alkaline stable protease for industrial use, an alkaline protease (protease BYA)was isolated from an alkalophilic Bacillus sp. Y, and its properties were characterized. Its optimumpHwas pH10.0-12.5, whencasein wasused as a substrate. In addition to the stability of protease BYAat pH 6.5-13.0, it was also very stable towards various surface-active agents, such as sodium dodecyl sulfate and sodium linear alkylbenzene sulfonate. Protease BYAwas most active at 70°C. The isoelectric point (p/) of protease BYAwas about 10.1. Protease BYAwas characterized as a serine protease because of its sensitivity to phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The protease seems to be related to proteases of the subtilisin family, such as subtilisin BPN', subtilisin Carlsberg, and No. 221 protease.
A number of studies on microbial alkaline
proteases have been done in view of structurefunction
relationships
and industrial
applica-
tions. Amongthem, alkaline proteases derived Bacilli1~5) and Strepto-
from alkalophilic myces,6) which
are known to be active
and
stable in highly alkaline conditions have drawn
serum albumin,
and ovalbumin
were purchased
from
Sigma Chemicals Co. (St Louis, Mo.). Hammerstein casein and keratin were obtained from E. Merck(Darmstadt, West Germany) and Tokyo Kasei Kogyo Co. (Tokyo), respectively. Oxidized insulin A and B chains, glucagon,
chymostatin, antipain, diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), and /?-chloromercuribenzoate (PCMB) were obtained from
Sigma. Streptomyces subtilisin inhibitor (SSI) was donated by Dr. Y. Mitsui. DEAE-53was a product ofWhatman Ltd. (Maidstone, Kent). Toyopearl HW-55 was provided tives, and to the fundamental questions of why by Toso Co., Ltd. (Tokyo). Pharmalyte 3-10 was obtained these proteases are so active and stable under from Pharmacia Japan (Tokyo). Dried yeast and soluble extreme conditions. starch were purchased from Wako Pure Chemicals (Osaka) and Junsei Chemicals Co., Ltd. (Tokyo), respectively. In this paper, we will describe the purification the attention of many researchers to their industrial use, especially as laundering addi-
and characterization from an alkalophilic
of an alkaline protease strain of Bacillus sp. Y,
which might have very useful characteristics for industrial
Bacterial strain. Alkalophilic Bacillus sp. Y strain screened from soil in an alkaline mediumwas used for the
application.
Materials
and Methods
Enzymes and chemicals. Subtilisin Carlsberg, subtilisin BPN', hemoglobin, myoglobin, chymotrypsinogen, bovine To whomcorrespondence should be addressed.
Cultivation and medium. Bacillus sp. Y. was grown aerobically in the culture medium described below at 35°C for 3 days. The culture medium contained 50g soluble
starch, 10g dried yeast, 1 g K2HPO4, 0.2g MgSO4à"7H2O, and 10g Na2CO3 per liter.
2252
H. Shimogaki et al.
Assay ofprotease
activity,
(a) Protease
activity
was
measured essentially by Anson-Hagiwara's method.8) To the reaction mixture (3ml) containing 0.6% casein and 10mM borax-NaOH buffer (pH 10.5), 0.5ml of enzyme solution was added at 35°C. After incubation for lOmin, the reaction was stopped by the addition of 3.2ml of stopper solution consisting of 0. 1 1 mtrichloroacetic acid, 0.22m sodium acetate, and 0.33m acetic acid (TCAmixture). The reaction mixture was kept at 35°C for lOmin, and filtered through Toyo filter paper No. 131. The absorbance of the filtrate was measured at 275 nm. One unit of alkaline protease activity (1 APU) was defined as the amount of enzyme liberating 1fj.g of tyrosine per min under the
conditions described to 100ml of distilled was vigorously stirred with ultrasonication
above, (b) Keratin (20g) was added water in a flask, and the suspension for 1 hr. The suspension was treated for 2min. To the reaction mixture
(3ml) containing 4% keratin and 10mM borax-NaOH buffer (pH 10.5), 0.5 ml ofenzyme solution was added with shaking (130rpm) at 35°C for 30min. Reaction was stopped by the addition of 3.2ml TCA-mixture as described above. The reaction mixture was filtered through a Toyo filter paper No. 131 and the absorbancy at 275nm of the filtrate was measured. Protein concentration. Protein concentration was measured by the method of Lowry et al.9) with bovine serum albumin as a standard. Polyaerylamide gel electrophoresis. SDS-PAGE was done by the method of Laemmli10) in a 12.5% polyacrylamide slab gel at pH 4.3. Proteins were stained
with 0.25% Coomassie brilliant Weber and Osborn.11*
The
blue by the method of
molecular
weight
of the
protease was estimated from the mobility of the protein band by the method of Andrew12) with bovine serum albumin, proteins.
ovalbumin,
Isoelectricfocusing.
and chymotrypsinogen
as marker
Isoelectric focusing was done on a
column (110ml, LKB Produkter) containing 1% Pharmalyte (pH range 3-10) with a linear density gradient of sucrose (0-50%) at 2°C for 72hr Vesterberg.13) After electrophoresis, fractionated into 1.5-ml portions,
by the method of the solution was and the pH was
terminal amino acids of protease BYAwere sequenced with an Applied Biosystems 377A protein sequencer by the method of Hewick et al.16) The phenylthiohydantoin (PTH)-amino acid derivatives were identified performance liquid chromatography (HPLC) attached to the sequencer.
by high system
Fractionation ofpeptides by HPLC.Fractionation of peptides by HPLCwas done on a Nihonseimitsu RP-18 column (7.5 x 250mm) equilibrated with 0.1% trifluoroacetic acid and eluted with a linear gradient of acetonitrile to reach
70%.
The flow rate
fractions
was 2ml/min.
The 2-ml
were collected.
Purification of protease. All purification procedures
were done at 4°C.
Step 1. Crude enzyme solution (2,650ml) was obtained by centrifugation of the culture broth of Bacillus sp. Y. (3,100ml)
at 10,000xg
for lOmin.
Setp 2. Solid ammoniumsulfate was added to the crude enzyme solution to 70%saturation and then the solution was stirred for 30min. The precipitate was collected by centrifugation at 14,000 x g, for 20min and dissolved in a minimal volume of 10mMsodium borate buffer (pH 9.3) containing 2 mMcalcium acetate. The enzymesolution was dialyzed against the same buffer overnight. Step 3. The dialyzed enzyme solution was put on a DEAEcellulose column (4.5 x 25cm) equilibrated with 10mMsodium borate buffer (pH 9.3). The column was washed with the same buffer. The enzymatic active fractions which were not adsorbed to the column were pooled and concentrated by ultra filtration using Diaflo cell a model 8,400 (Amicon). Step 4. The concentrated enzyme solution was put on a Toyopearl HW-55 column (1.6 x 90 cm) equilibrated with 20mM Tris-HCl buffer (pH 7.2) containing 2mM calcium acetate and 0.2m NaCl. The column was eluted with the same buffer at a flow rate of20 ml/hr. The enzymatic active fractions were combined and brought to 70% saturation by the addition of solid ammoniumsulfate. Step 5. The precipitated protein was collected by centrifugation and dissolved in a minimal volume of20 mM Tris-HCl buffer (pH 7.2) containing 2 mMcalcium acetate.
The enzymesolution was dialyzed against the samebuffer overnight and then stored at -20°C. The preparation thus obtained is here after designated protease BYA.
measured at 2°C. Protease activity was assayed by the
assay method (a) described above. Amino acid analysis. The amino acid composition of protein was analysed by the method of Spackman et al.14) using a JEOL amino acid analyzer, JLC 200A (Nihon-
Results
Purification ofprotease B YA The alkaline protease BYA was purified denshi, Tokyo). Protein was hydrolyzed with 5.7n HC1 from the crude enzyme solution through 5 in evacuated and sealed tubes at 110°C for 24-72hr. steps of purification including salting out, Tryptophan was measured by the method of Pajot.15) anion exchange chromatography, and gel N-Terminal amino acid sequence analysis. The Nfiltration to a single band on SDS-PAGE. The
Surface-active Table I. Purification
c
1.
+ SteP§
Supernatant
of culture fluid 2. Ammonium sulfate fractionation 3.
Anion
exchange chromatography 4. Gel filtration 5. Ammonium sulfate fractionation
Total activity (APUx
Agent- and Alkaline-resistant
of Protease .
P"
Bacillus Protease
2253
BYA
Specific activity (APU/mg
Yield (%)
10-6)
(mgj
x10-3)
6.6
3450
1.9
100
5.9
1280
4.4
90
4.8
400
ll.8
72
3.8 3.4
273 216
13.8 15.9
57 52
Fig. 2. Effects of pH on Enzyme Activity. Enzymatic activity was measured in 20 mMbuffer contain-
ing 0.6% casein as substrate at 35°C. The buffers used were sodium acetate buffer for pH 4.0-5.5, sodium
phosphate buffer for pH 6.0-7.5, 7.5-8.5,
glycine-NaOH
buffer
Tris-HCl buffer for pH
for
pH
8.5-9.5,
borax-
NaOHbuffer for pH 9.5-1 1.0, sodium phosphate-NaOH buffer for pH ll.0-12.0, and KCl-NaOH for pH 12.013.0. #, protease BYA; O, subtilisin Carlsberg.
pi
Results
of isoelectric
focusing
of protease
BYAindicated that the pi of protease BYA
was about 10.1, and was higher than those of subtilisin shown).
Fig.
1.
SDS-PAGE.
Carlsberg
and BPN'17)
(data
not
Effects of pH on enzymatic activity
The effects of pH on caseinolytic activity Lane 1, protease BYAdenatured in 0.1% SDS at 100°C for 2min; lane 2, protease BYAtreated with 1mMDFP were measured at 35°C. Protease BYAwas before to heat denaturation in 0.1% SDS at 100°C; lane most active at the broad range of pH 10.0-12.5 3, standard proteins bovine serum albumin, ovalbumin, (Fig. 2). The optimum pH is about 1.5 pH units and chymotrypsinogen.
higher than that of subtilisin Carlsberg, and comparable with Bacillus No. 221 protease.1}
specific activity of the purified protease BYA
rate and Effects of pH on the stability ofprotease BYA yield of protease BYAfrom the crude enzyme The effects of pH on the stability ofprotease solution were 8-fold and 52%, respectively. The BYAwere measured at 25°C and pH 4-13. purification procedures are summarized in After incubation at a given pH for each sample Table I. for 24 hr, the enzymatic activity was measured at pH 10.5. Protease BYAwas stable at pH Molecular weight ofprotease BYA 6-13 under the described conditions, while The molecular weight estimated by gel subtilisin Carlsberg showed 80 and 20% of its filtration using Toyopearl HW-55was approx- activity (pH 10.5), at pH 12 and 13, respectively. was 15,900 APU/mg. The purification
imately 21,000 (data not shown). However, the
molecular weight by SDS-PAGE after treatment with 1mM DFP was approximately 42,000 (Fig. 1).
Effects of temperature on enzymaticactivity Protease BYAwas most active at 70°C, which was about 10°C higher than the optimum
2254
H. Shimogaki et al.
of subtilisin Carlsberg. About 50 and 40% of the protease BYAactivity remained even after incubation at 75 and 80°C for lOmin, respectively. Under the latter condition, subtilisin Carlsberg lost its activity (data not shown). The stability was slightly higher than that of Bacillus No. 221 protease.1} Effects of temperature on the stability ase BYA Protease BYA was incubated
ofproteat various
Effects of various divalent cations on protease BYAactivity The effects of divalent cations on protease BYAactivity were tested at a 1 mMconcentration of metal ions in 20niM borax-NaOH buffer (pH 10.5) at 40°C for 24hr. Protease
BYAwas markedly inhibited CuSO4 (98% and 74% inhibition,
by HgCl2 and respectively)
and moderately by CdCl2 (25% inhibition), not by CoCl2, NiCl2,
but
BaCl2, FeSO4, CaCl2,
MgSO4, MnCl2, or ZnCl2.
temperatures and pH 10.5 for lOmin, and the residual activity was measured. Protease BYA Effects of various denaturants and surface-active was stable at temperatures up to 55°C and agents on protease BYAactivity showed about 90% and 30% of the native
activity at 60°C and 70°C, respectively, where subtilisin Carlsberg lost its enzymatic activity. Effects of inhibitors The effects
of several
inhibitors
on the
Protease BYA and subtilisin Carlsberg (1,000 APU) were incubated in various denaturants such as 0.1n NaOH and 6m
guanidine-HCl (pH 10.5) at 35°C for 30min, then their residual activities were measured. Protease BYA retained 90 and 84% of its
activity of protease BYAwere examined by activity in the presence of denaturants, NaOH However, measuring residual activity after incubation of and guanidine-HCl, respectively. the enzyme with inhibitors
in 50mMTris-HCl
buffer (pH 7.0) at 35°C for 30min. Protease BYA was completely
inhibited
by active
subtilisin Carlsberg showed approximately 0 and 16% of its original activity, with these denaturants, respectively. From those data,
site-directed inhibitors of serine protease, such protease BYAseems to be more stable against as DFP and PMSF, but not by EDTAand PCMB (Table
II),
indicating
that
protease
BYAis a serine protease. Protease BYAwas markedly inhibited by SSI.18)
Table III. The Stability of Protease BYAand Subtilisin Carlsberg in the Presence of Various Surface-Active Agents at pH 10.5
Residual activity (%) on
Surface-active
activity
Sodium linear alkylbenzene sulfonate (C = 10-14) Sodium-olefine sulfonate (C=14-18, p=3) Sodium-alkylpoly (oxyethylene) sulfonate (C=11-13, p=3)
Table II. Effects of Several Inhibitors Protease BYAActivity Inhibitor None EDTA DFP PMSF PCMB
Antipain Chymostatin SSI
Concentration
5 mM 1 him 1 him 1 him
1 00 /ig/ml 1 00 /^g/ml 5 fiM
Residual (%)
100 97 0 0 122 124 77 2
Protease BYA
(C=9,
93
p=9.5)
Alkylethoxylate
83 81
(C= 1 1-13, p= 9)
(1,000APU/ml)
Subtilisin Carlsberg
84
Sodium dodecyl sulfate Nonylphenylethoxylate
Proteases
The reaction mixture consisting of enzyme (1,000 APU), and inhibitor at given concentration in 50mM
agent (5% v/v)
91
were incubated
in 50mM
borax-NaOH buffer (pH 10.5) containing surface-active
agents at 40°C for 4hr, and then residual activities were Tris-HCl buffer (pH 7.0) was incubated at 35°C for 30min. measured by method (b) in Material and Methods. C, distribution of carbon chain length; p=polymerization The residual activity was assayed by method (a) described in Materials and Methods. degree.
Surface-active
Agent- and Alkaline-resistant
these denaturants than does subtilisin berg under these
conditions.
Similar
Bacillus Protease
2255
Carls- The results showed that protease BYAwas
experi-
generally
more stable than subtilisin
Carlsberg
ments were done in the presence of 5% (v/v) of under these conditions. The difference in their was marked in the case of use of various surface-active agents activities at 40°C andwerepH stabilities 10.5 for 4hr. The residual sodium linear alkylbenzenesulfonate, sodium dodecyl sulfate, and nonylphenylethoxylate.
measuredwith keratin as the substrate, because of inaccuracy of the assay method (a) in the These results indicate that protease BYAis a presence of surface-active
agents (Table
III).
surface-active
agent-resistant
protease.
Fig. 3. Cleavage of Oxidized Insulin A and B Chains and Glucagon with Protease BYA. To the substrate (1 mg) in lml of 2mM borax-NaOH buffer (pH 10.0), 160-1,600APU of protease BYA was added. Thereaction mixture was incubated for an appropriate time, then the reaction was stopped by the addition of 55/il of 1.8% trifluoroacetic acid. The cleavage products were separated by HPLC.(a) Cleavage of oxidized insulin A chain with protease BYA. (b) Cleavage of oxidized insulin B chain with protease BYA. (c) Cleavage of glucagon with protease BYA. Perpendicular arrows indicate split bonds. Heavy and light arrows indicate major and minor site ofcleavage, respectively. The major and minor cleavage mean 100 and less than 40%hydrolysis. The results of similar experiments with subtilisin BPN' and Carlsberg are also included for comparison.17'22)
2256
H. Shimogaki et al.
Substrate specificity
Carlsberg.
Substrate specificity of protease BYAwas studied with oxidized insulin A and B chains, Amino acid composition and glucagon
as substrates.
These substrates
were digested with protease BYA,and samples were drawn out at appropriate
intervals.
The
digestion products were separated by HPLC. Fromthe amino acid analysis and measurement of each peptide separated, the rate and locations of cleavage sites were estimated (Fig. 3). In the case of oxidized insulin A chain, the Glu17-Asn18
bond
is the
most susceptible
followed by the Ser12-Leu13 bond, to protease
BYA.Other bonds are rather resistant to this enzyme. When we used oxidized insulin B chain
as a substrate, the Tyr26-Thr27 bond was hydrolyzed most rapidly, followed by the
Carlsberg,20)
and No. 221 protease1} are in-
cluded for comparison. In comparison with
that of the three knownBacillus enzymes, the phenylalanine content of protease BYAwas very high, and the arginine content was a little higher than that of No. 221 protease, and
higher than those of two subtilisins. On the other hand, serine and valine contents were
both lower than in the Bacillus proteases. N-Terminal amino acid sequence The N-terminal
Gln4-His5 bound. The other bonds susceptible to protease Glu13-Ala14,
The amino acid composition of protease BYAis shown in Table IV. The amino acid composition of subtilisin BPN',19) subtilisin
BYA were, Leu6-Cys7-SO3H, and Tyr16-Leu17. In the case of
amino acid
sequence
of
DFP-treated protease BYAwas identified Edman degradation
by
up to 21 amino acid
glucagon as a substrate, the Leu26 -Met27 bond was most susceptible followed by the Phe6Thr7, and Leu14-Asp15 bonds, which were
residues (Fig. 4). The N-terminal amino acid of protease BYAwas asparagine and differed
completely hydrolyzed. In addition to these bonds, Ser^-Lys^, Gln20-Asp21, and Val23-
Table IV. AminoAcid CompositionofProtease
Gln24 bonds were partially
hydrolyzed under
these experimental conditions. The results of similar experiments using subtilisin BPN' and subtilisin Carlsberg towards an oxidized insulin B chain and glucagon20)
from those of the two subtilisins
are also included
these three enzymes. However, in the case of subtilisins, there are more cleavage sites than in protease BYA. In the same way, cleavage
points in glucagon for protease BYAwere markedly fewer than for subtilisin Carlsberg. These results indicate that the two subtilisins cleave these substrates more non-specificially
than does protease BYA. Immunological properties ofprotrease B YA Antiserum against BYAinhibited protease BYAactivity, but not that of subtilisins BPN' and Carlsberg (data not shown). The results
show that protease BYA is immunologically distinguishable from subtilisins BPN' and
BYA
Subtilisin
Ammoacid PrR°'efe
BPN"»> Carlsberg*» BYA
in
Fig. 3 for comparison. From this figure, it can be seen that the cleavage of the Gln4-His5 bond in the oxidized insulin B chain is commonfor
and No. 221
No' 22\.
& protease"
mol (%) Trp Lys His Arg Asx Thr Ser Glx Pro Gly Ala
l /2Cys
Val Met He Leu Tyr Phe
1.4 3.4 2.2 3.9 15.0 6.9 7.8 6.3 5.5 ll.8 12.8 0 5.5 1.4 3.4 6.1 4.1 3.2
1.1 4.0 2.2 0.7
10.2 4.7 13.5 5.5 5.1 12.0 13.0 0 10.9 1.8 4.7 5.4 3.6 1.1
0.4 3.3 1.8 1.5 10.2 6.9 ll.6 4.4 3.2 12.7 14.9 0 ll.3 1.8 3.6 5.8 4.7 1.4
1.8 2.1 2.8 2.8 10.2 6.3 8.1 5.6 5.6 13.7 15.8 0 9.5 1.4 3.2 7.7 2.5 0.7
Aminoacid contents of Ser and Thr were obtained by extrapolating the observed value to zero time ofhydrolysis.
Surface-active P rotease
Agent- and Alkaline-resistant
BYA
Np|v|arJGi|--|VKAP]vi&QN|NyJgJly|§|--
Subtilisin
BPN'
AQS§PY§VSQI@P-§LHSQ§YT§--
Subtilisin
Carlsberg
AQT@PY^PLlgADJKvgA-Q§FK§--
No. 221
2257
Bacillus Protease
Protease
AQS@PW^l]sR@QgPAgHg-RJG|--
Fig. 4. N-Terminal Amino Acid Sequences of Protease BYA, Subtilisin BPN',17) Subtilisin and No. 221 Protease.21} The commonsequences with those of three protease of subtilisin family are boxed.
protease (alanine).17'21) those
of subtilisin-type
Whencompared with
proteases,
the
N-
about 21,000, which is different obtained
by SDS-PAGE,
(about
Carlsberg,17)
from the value 42,000).
A
terminal sequence was homologous with pro-
similar phenomenonwas also observed in the case of an alkaline protease derived from
Carlsberg17 (9 out of21 residues), and Bacillus No. 221 protease21 (8 out of 20 residues).
Bacillus AH101.5) Wehave to wait for further structural analysis ofprotease BYAto measure
tease BPN' (7 out of 21 residues),
subtilisin
Since protease BYAis a serine protease and is inhibited by SSI, it is possible that protease
Discussion
Protease BYAisolated from Bacillus sp. Y.
strain
is a surface-active
the molecular weight.
agent- and alkaline-
resistant serine protease. Protease BYAis most active at pH 10.0-12.5. Its optimal pH is higher than for the known subtilisins, and is comparable to those of alkalophilic Bacillus AH1015) and Bacillus sp. No. 221 protease.1} However, the pH-activity curve measured at optimal pH was somewhat broader than for the latter two proteases. The pH stability is very similar to that of a protease from alkalophilic Bacillus sp. GX6638 reported by Durham.17) However, we are not able to compare it with those of two proteases from alkalophilic Bacillus AH101 and No. 221 directly, because of the difference in experi-
BYAis a memberof the subtilisin family. The homologous amino acid sequences between subtilisin BPN', subtilisin Carlsberg, and No. 221 protease also suggest this possibility. Therefore, protease BYAmight be a very interesting model to investigate the origin of its alkaline
stability
and surface-active
agent-
resistant nature from the standpoint of protein chemistry. The results of cleavage of three peptides, oxidized A and B chains of insulin, and
glucagon showedthat protease BYAseems to have no strict amino acid specificity. Whenwe compare the site ofcleavage of insulin B chain
with protease BYAto those with three other Bacillus proteases, cleavage sites with protease
mental conditions. Although Bacillus sp. Y BYA are somewhat similar to those with strain is not a thermophilic bacterial type, the subtilisin Novo,22) but are fewer than in the optimal temperature (70°C) and heat stability case of the three Bacillus proteases. Therefore,
of protease BYAindicated that protease BYA is more heat-stable than subtilisins BPN' and Carlsberg. In addition to the unique nature of
in respect to specificity, protease BYA is less non-specific than the subtilisins, which means that protease BYA cleaves these peptides at
protease BYAdiscussed above, the protease is
manyfewer points. Although we do not yet
moreresistant to various surface-active agents than is subtilisin Carlsberg. The molecular weight by gel filtration was
know the rules governing
cleavage
sites,
the
presence of a hydrophobic amino acid at least at one or two ofS2, Sx and S_x sites appears
2258
H. Shimogaki et al.
to be essential. Acknowledgment.
The
authors
thank
Professor
Y.
J. Randall, /. Biol. Chem., 193, 265 (1951). U. K. Laemmli, Nature, 227, 689 (1970). P. Weber and M. Osborn, J. Biol. Chem., 244, 4406 (1969).
Mitsui of the Faculty of Engineering, Nagaoka University of Technology for his kind donatrion of SSI, and Miss S. Mine for her technical assistance in the HPLCexperiments.
P. Andrew, Biochem.
preparing the manuscript.
D. H. Spackman, W. H. Stein and S. Moore, Anal.
The authors
thank
Mrs. Julia
Thile
for her advice
in
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