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Isolation of isoinhibitors of trypsin from porcine colostrum
836
BIOCHIMICA ET BIOPHYSICAACTA
BBA 35767 ISOLATION OF ISOINHIBITORS OF TRYPSIN FROM PORCINE COLOSTRUM
L A W R E N C E F. KRESS, SUSAN R. MARTIN* AND M. LASKOWSKI, SR.
Laboratory of Enzymology, Roswell Park Memomat Inst,tute, and the Department of Biochemzstry, Roswell Park Dwzsion, State Universzty of New York at Buffalo, Buffalo, N.Y. 14203 (U.S.A .) (Received September i4th, i97o)
SUMMARY
With the aid of chromatography on CM-cellulose the trypsin inhibitor from swine colostrum was subfractionated into four isoinhibitors. Each isoinhibitor migrated as a single band in disc electrophoresis at pH 4.3, and had the same specific activity against trypsin. All isoinhibitors contained carbohydrate. Only small differences in composition of isoinhibitors were detected. Sialic acid was present in one of them, and its removal did not alter inhibitor activity. The isoinhibitors are active against bovine and porcine trypsin, a-chymotrypsin and chymotrypsin B. They are inactive against carboxypeptidase B and the milk clotting activity of pepsin at pH 4.9. The reactive site of the inhibitor involves a lysine-X bond.
INTRODUCTION
Several years ago 1, a trypsin inhibitor was discovered in swine colostrum. It was isolated in a crystalline form, but showed some heterogeneity when subjected to electrophoresis. Recently, MARTINs devised a more efficient method of purification of the inhibitor. The preparation contained carbohydrate (II%) and was heterogeneous by disc electrophoresis. This preparation served as a starting material for the separation of individual isoinhibitors, using equilibrium chromatography and disc electrophoresis as criteria of purity. The purification, composition, and some properties of the isoinhibitors are now reported. MATERIALS AND METHODS
Bovine trypsin (twice crystallized), carboxypeptidases A and B (DFP treated), a-chymotrypsin and pepsin were obtained from Worthington. Porcine trypsin was from Novo Industry. Chymotrypsin B was prepared in this laboratory 3. BenzoylL-arginine ethyl ester hydrochloride was from Mann. Glucosamine and galactosamine Present address : Department of Biology, State University of New York at Buffalo.
BiochiA. Biophys. Acta, 229 (I97I) 836--844
TRYPSIN INHIBITORS FROM PORCINE COLOSTRUM
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were from Pfanstiehl Laboratories, Inc. CM-cellulose was purchased from Schleicher and Schuell. Microgranular celluloses, CM-52 and DE-52, preswollen, were obtained from H. Reeve Angel and Co. Sephadex G-5 o was from Pharmacia. Porcine colostrum was collected for periods up to 36 h post partum. The samples were frozen as soon as possible and stored frozen until used.
Assays Trypsin and trypsin inhibitory activities were determined by the method of SCHWERT A N D TAKENAKA4, as modified by KASSELL et al. 5. Specific inhibitory activity was expressed as/zg of trypsin inhibited per I.O A2s0 nm unit of inhibitor*. Analytical methods Amino acid analyses were performed according to MOORE AND STEINs using a Beckman-Spinco I2oB analyzer. Approx. 3-mg samples of inhibitor were hydrolyzed in 8 ml of 5.7 M HC1 for 48 h at IIO °. Cystine was determined as cysteic acid 7. Amino sugars were determined b y hydrolyzing 2-mg samples in I.O ml of 4 M HC1 for 4 h at IOO°. Samples (0.2 ml) were applied directly to a 0. 9 cm × 15 cm column of the analyzer and eluted with p H 5.28 buffer. Neutral sugars were determined by gasliquid chromatography according to the method of LENHARDT AND WINZLER8. The authors are grateful to Dr. R. J. Winzler and to Mr. John Bleecher for the analysis. Sialic acid was determined by the thiobarbituric acid test described by CASSIDY et al. 9. Free sulthydryl was estimated according to ELLMANN10. Disc gel electrophoresis was performed by the method of DAVIS11 at p H 9.5 and according to REISFELD et al. 12at p H 4.3. The authors are indebted to Dr. Sally Schneider for these experiments. Carboxypeptidase determinations Approx. 3.o-mg samples of inhibitor dissolved in o.oi M sodium borate-o.i M NaC1 (pH 8.0) were incubated with 0.095 mg of carboxypeptidase B at 23 °. At suitable intervals a o.I-ml aliquot of the reaction was pipetted into 0. 9 ml of the p H 2.2 amino acid analyzer buffer and frozen until analysis. For carboxypeptidase A analysis, 4.9 mg of inhibitor (Peak II, Fig. 2) was dissolved in 0. 4 ml of 0.2 M LiC1. Carboxypeptidase A (o.13 mg) was added, the p H adjusted to 8.2, and the reaction allowed to proceed at 23 °. Aliquots (o.I ml) were withdrawn, pipetted into 0. 9 ml of p H 2.2 diluting buffer and frozen until analyzed. RESULTS
Purification of trypsin inhibitor Table I summarizes the results for a typical batch of colostrum. Step L Swine colostrum was thawed and centrifuged at 18 ooo x g for I.O h at o °. The fat layer at the top, and the residual pellet at the bottom were devoid of activity and were discarded. Step 2. To the liquid, an equal volume of 5 % trichloroacetic acid was added slowly, and stirring was continued for 2 h at room temperature. The precipitate was * P r o t e i n w a s e x p r e s s e d in A2sonm units. One u n i t refers t o t h e a m o u n t which, i f di s s ol ve d in i ml, w o u l d h a v e a n a b s o r b a n c e of u n i t y a t 28o rim.
Biochzm. Bzophys. Acta, 229 (1971) 836-844
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L. F. KRESS
et al.
TABLE I SUMMARY OF PURIFICATION
PROCEDURE
Purification step
Total *nh*bitor (units X
R a w colostrum after centrifugation Trichloroacetic acid fractionation (NH4)=SO 4 fractionation CM-cellulose c h r o m a t o g r a p h y Sephadex G-5 o Gradient c h r o m a t o g r a p h y on microg r a n u l a r CM-cellulose
% Recovery Specific of inhibitory inhzb~tory act*vity activzty
10 6)
5.86 3.i8 3.7 ° 3.31 2.42
ioo 54.3 63.2 56.5 41.3
--
--
44oo 4815 575 ° 6o00
filtered on W h a t m a n No. 50 filter paper. The filter cake was re-extracted twice, with 2.5% trichloroacetic acid, and discarded. Step 3. Solid (NH4)2SO * was added to the combined filtrates to attain 80% saturation (603 g/l). After standing overnight at room temperature, the precipitate was collected on W h a t m a n No. 50 filter paper, dissolved in o.oi M ammonium formate (pH 3.7) and dialyzed against the same buffer at 2 °. The slightly turbid solution was clarified by centrifugation. Step 4. The inhibitor was charged on a 4 cm × 60 cm CM-52 cellulose column. The first peak was discarded. The starting buffer was replaced with buffer containing 0.3 M NaC1, A second peak contained 75 % of the inhibitory activity. It was pooled, dialyzed against distilled water, lyophilized, and stored at --18 °. Step 5. The active material was dissolved in o.oi M HCI-o. 3 M NaC1 (pH 2.1) and passed through a 5 cm × IOO cm column of Sephadex G-5o equilibrated with the same solution. The breakthrough peak contained no activity. Tubes which had a specific activity greater than 5500 units/A2s0 nm unit were pooled, concentrated, 0.350 0.300 t,t
0.2 z_
0
~0.Z00
O
tD +
0.100
ao
60
ioo
t4o
teo
TUBE No.
Fig. I. G r a d i e n t c h r o m a t o g r a p h y of swine colostrum t r y p s i n inhibitor on m i c r o g r a n u l a r CMcellulose. Swine eolostrum inhibitor (91 A2s0 nm units) was dissolved in 15 ml of o.oi M sodium succinate (pH 5.5) and dialyzed against the same buffer for 17 h. The solution was placed on a 1.5 cm × 88 cm column of m i c r o g r a n u l a r CM-cellulose. The protein was eluted w i t h a linear gradient f r o m o.oi succinate (i.o 1) to o.oi succinate-o.2 M NaC1 (i.o 1). Flow rate was 3 ° ml/h, and 5-ml fractions were collected. The average specific activity of Peak I was 3000 units/A 280 nm unit; t h a t of Peaks I I - V averaged 60o0 units/A=s8 nm u n i t in three separate c h r o m a t o g r a p h i c runs. , A 2 s 0 nm; O - - C ) , m o l a r i t y (suceinate + NaCI); [ [, t u b e s pooled.
B,och,m. Biophys. Acta, 229 (1971) 836-844
839
TRYPSIN INHIBITORS FROM PORCINE COLOSTRUM 0.350
0.350 -
0.300
0.300
~
oo200
0.200
i
,g
~t
0300
2O
40
60
80
~00
20
40
60
80
100
TUBE N O .
TUBE N o .
0.350 0.300
0.35c 0.30C
00.200 00.20C
0.J00 O.;OC
20 TUBE No.
40 60 80 TUBE N O .
100
Fig. 2. E q u i l i b r i u m c h r o m a t o g r a p h y of individual swine colostrum t r y p s i n inhibitors on microg r a n u l a r C1Vi-cellulose. I n all the above figures, the R o m a n n u m e r a l s correspond to those used in Fig. I. E a c h inhibitor was dissolved in IO ml of the respective buffer to be used and dialyzed against t h a t buffer. The protein solutions were placed on a 1,5 cm × 88 cm c o l u m n of microg r a n u l a r CM-cellulose and eluted w i t h the following buffers: Peak I I (22.8 A,80 nm units) : o.oi M s o d i u m succinate (pH 5.5); Peak I I I (35.9 -4,80 nm units): o.oi M succinate-o.oi 5 M NaC1; Peak I V (24.2 A,80 nm units): o.oi M succinate-o.o3 o M NaC1; Peak V (27. 5 A280 nmunits) : o.oi M succinate-o.o45 M NaC1. The flow rates were 3 ° ml/h, and 5-ml fractions were collected. The void v o l u m e of the columns occurred at T u b e io.
adjusted to p H 4.5 and dialyzed against distilled water. The material was lyophilized and stored at --18 °. Disc electrophoresis revealed at least six bands. Specific activity was 5750 units/A,80 nm unit. Step 6. The accumulated material (usually IOO A280 nm units) was dissolved in o.oi M sodium succinate (pH 5.5). The solution was dialyzed, charged on a column of CM-52, and eluted with a linear gradient of o.oi M sodium succinate (pH 5.5) and o.oi M sodium succinate-o.2 M NaC1 (pH 5.5). The results of a typical experiment are shown in Fig. I. The protein fractions were assigned Roman numerals. Fraction I consisted of heterogeneous material of low specific activity. Peaks II, I I I , IV, and V all had activity approaching 6000 units/A 2s0 nm unit. Recovery amounted to about 9 ° %. The peak tubes were pooled, concentrated, and stored at 4 ° . Material from five separate experiments was combined, dialyzed against water, and lyophilized. Step 7. Material from each peak obtained from the previous step was subjected to equilibrium chromatography on CM-52. NaC1 concentrations were so chosen that the inhibitor peak would emerge only after at least three void volumes had passed through the column. The column and the inhibitor solution were carefully equilibrated with the buffer used, prior to chromatography. The results are shown in Biochim. Biophys. Acta, 229 (I971) 836-844
840
L.F. KRESS et al.
Fig. 3. P o l y a c r y l a m i d e gel electrophoresis of swine c o l o s t r u m t r y p s i n inhibitors. A p p r o x . 425 # g of t h e inhibitor fraction purified b y e q u i l i b r i u m c h r o m a t o g r a p h y were applied to a 1 5 % acryla m i d e s e p a r a t i n g gel a t p H 4.3. Electrophoresis was p e r f o r m e d at 5 m A per gel for 16o rain. T h e arrow m a r k s t h e position of t h e t r a c k i n g d y e ( b r o m p h e n o l blue) at t h e e n d of t h e run. T h e gels were s t a i n e d in 1 % aniline blue-black a n d d e - s t a i n e d b y leaching w i t h IO°//.o acetic acid. T h e R o m a n n u m e r a l s d e s i g n a t i n g t h e fractions correspond to t h o s e u s e d in Fig. I.
0.120
®
®
0.090 0~0.0£0 ....ii 0"5
0.030
20 40
IO
80I
I
I00
O
I
120 140 160 180 200
TUBE No. Fig. 4. Purification of P e a k I I I i n h i b i t o r o n CM-cellulose. P e a k I I I i n h i b i t o r (see Fig. 2) w a s dissolved in 5.5 ml o f o.oi M s o d i u m s u c c i n a t e - o . o i 5 M NaC1-6 M u r e a (pH 5.15) a n d placed on a 1.5 c m × 88 c m c o l u m n of m i c r o g r a n u l a r CM-cellulose e q u i l i b r a t e d w i t h t h e s a m e buffer. T h e initial h e t e r o g e n e o u s p e a k lacked i n h i b i t o r a c t i v i t y . T h e arrow i n d i c a t e s t h e p o i n t at w h i c h a linear g r a d i e n t of o.o15-o. 5 M NaC1 in o.oi M s u c c i n a t e - 6 M u r e a (pH 5.15) was initiated. T h e disc electrophoresis p a t t e r n of t h e m a t e r i a l w h i c h e m e r g e d is s h o w n a b o v e t h e peak. T h e inhib i t o r y m a t e r i a l h a d a specific a c t i v i t y of 585 ° units]A280 am unit. - - , A2s0 nm; , M NaC1.
Biochim. B,ophys. ,4eta, 229 (1971) 836-844
TRYPSIN INHIBITORS FROM PORCINE COLOSTRUM
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Fig. 2. All of the main peaks were well resolved and chromatographed symmetrically. The material from each peak was pooled, concentrated by rotary evaporation, dialyzed against distilled water, and lyophilized. This lyophilized material was used for analyses.
Disc electrophoresis The patterns from disc electrophoresis at pH 4-3 of the inhibitor peaks are shown in Fig. 3. All the inhibitor peaks gave single bands except Peak III which appeared as a double band. Peak I was heterogeneous as expected. Since Peak III exhibited two bands in both the pH 4.3 and pH 9.5 electrophoresis systems, it was subjected to further purification. Equilibrium chromatography of Peak III under the conditions used for Peaks II and IV showed that the material was not a mixture of Peaks II and IV. The successful purification of Peak III is illustrated in Fig. 4- Gradient chromatography at pH 5.1 in the presence of 6 M urea resulted in the removal of inactive material followed by the appearance of an active peak which migrated as a single band in disc electrophoresis. TABLE II AMINO ACID AND CARBOHYDRATE COMPOSITION OF INHIBITORS ISOLATED FROM SWINE COLOSTRUM Results are expressed as residues per molecule based on a value of isoleucine = i.oo.
Component
Peak I
Peak I I
Peak I I I
Peak I V
Peak V
Lysine Arginine Aspartic acid Threonine* Serine* Glutamic acid Proline Glycine Alanine Cystine** Valine Methionine*'* Isoleucine Leucine Tyrosine Phenylalanine
1.72 5.Ol 9.49 3.84 5.26 18.95 19.74 12.83 5 .°1 6 2.53 o.59 (i.oo) 4.3 ° 2.39 2.99
1.23 3.56 5.81 4.27 2.21 5.84 6.46 4.66 5.05 6.19 1.98 o.71 (i.oo) 4.09 2.44 3.76
2. io 3.72 6.08 4.31 2.22 6.13 6.79 4.85 5.18 6 2.2o 0.74 (i.oo) 4 .28 2.54 3.69
2.17 3.68 6.o9 4.25 2.14 5.88 6.71 4.80 5.15 6 2.38 o.81 (i.oo) 4 .18 2.50 3.61
1.97 3.8o 6.02 4.23 2.o8 5.66 6.92 4.91 5.29 6 2. i i o.82 (i.oo) 3.98 2.55 3.28
Glucosamine Galactosamine Sialic acid
1.94 o.32 o.61
3.7 ° o.39 0.80
3.74 o.46 o.oo
3.82 o.41 0.06
3.29 o.35 o.oi
Fucose Mannose Galactose Glucose
0.23 1.44 1.o6 o.o7
0.24 1.85 I.O6 o.04
0.38 2.22 1.41 Trace
0.42 1,79 1.42 o
0.38 2.o 7 i. 19 o.95
* N o t corrected for decomposition d u r i n g hydrolysis. ~* D e t e r m i n e d as cysteic acid in performic acid-oxidized Peak II. The values for the o t h e r peaks were a s s u m e d to be t h e same, since the recoveries of half-cystine were the s a m e for all peaks. "'* Uncorrected for decomposition; however, the value for methionine sulfone in performic acid-oxidized Peak I I was o.94 residue.
Biochim. Biophys. Acta, 229 (1971) 836-844
842
L.F. KRESS et al.
Amino acid analyses The results of amino acid analysis are shown in Table II. The Roman numerals correspond to the peaks in Fig. I. Fraction I had a composition quite different from the other four. Cystine was determined on Peak I I as cysteic acid. The value for Peak I I was taken as the value for all the peaks, since the recovery of half-cystine in acid hydrolysates had been the same for all the peaks. All the values were corrected for 9% moisture. There were no differences in the amino acid composition of Peaks II, III, IV, and V, except for the lack of a lysine in Peak II. This was confirmed by the carboxypeptidase determinations (see below). Histidine and tryptophan (determined spectrophotometrically 13) were absent. Carbohydrate analysis Table I I shows that all peaks contained four glucosamine residues and only a trace of galactosamine. Only Isoinhibitor I I contained sialic acid, and no change in activity was noted after removing it with neuraminidase. All the sialic acid was shown to be the N-acetyl form in the butanol-propanol-HC1 chromatography system of SVENNERHOLM AND SVENNERHOLM14. The results of gas-liquid chromatography determinations of neutral sugars are also shown in Table II. The only significant difference among the active peaks is the presence of 0.95 residue of glucose in Peak V. C-terminal amino acid Incubation with carboxypeptidase B resulted in the rapid release of one residue of lysine from Isoinhibitors 11I, IV, and V. No significant amount of lysine was released from Isoinhibitor II, even after 6 h. On the other hand, exposure of Peak I I to carboxypeptidase A resulted in the slow release of threonine (o.53 residue in 21 h). No other amino acids were released by either of the carboxypeptidases in any of the inhibitor peaks. It was concluded that Isoinhibitors I l l to V all had a carboxyterminal lysine. Isoinhibitor I I differed from the others by one less lysine, and its C-terminal amino acid was threonine. N-terminal The dansyl chloride technique of GRAY15 and the cyanate procedure of STARK 16 were performed on both the intact and performic acid-oxidized inhibitor. All these methods failed to reveal the amino terminal residue, which is not unusual with glycoproteins 1~. Free sulfhydryl determination Treatment with the ELLMANN10 reagent indicated absence of free sulfnydryl groups, which led to the conclusion that the inhibitor possesses three disulfide bonds (Table II). Reactive site Colostrum inhibitor (3.0 mg of Peak I I material) was incubated with o.8 mg of porcine trypsin in 0.75 ml of o.ooi M HCI-o.o2 M CaC12 (pH 3.4) at 23 °. After 96 h the p H was adjusted to 8.0 with 0.2 M borate (pH 8.0) containing I.O M NaC1. Carboxypeptidase B (o.II mg in 30 #1) was added, and the reaction continued for 72 h. The p H was then adjusted to 2.o with 0.2 M HC1, and the reaction mixture Biochim. Biophys. ,4cta. 229 (1971) 836--844
TRYPSIN INHIBITORS FROM PORCINE COLOSTRUM
843
frozen for amino acid analysis. The blank consisted of 0.8 mg of trypsin incubated at pH 3.4 for 9 6 h at which time the pH was adjusted to 8.0, and 3.0 mg of Peak II inhibitor and o.II mg of carboxypeptidase B were added. After 72 h the mixture was adjusted to pH 2.0. A net release of o.II #mole of lysine was observed in the experimental over the control tube while no arginine was detected. The amount of lysine recovered was 33% of that expected for full conversion to modified inhibitor. The actual net amount of lysine released from the inhibitor may be even higher, since the blank was set up to correct for the maximum possible release of lysine or arginine from tryptic autolysis products. It was concluded that the reactive site of the swine colostrum inhibitor involves a lysine-X bond.
Inhibitory activity with other proteolytic enzymes Swine colostrum inhibitor is also active against a-chymotrypsin, chymotrypsin B, and porcine trypsin. Removal of the sialic acid residue does not affect inhibitory activity in any of the above cases. The colostrum inhibitor does not inactivate carboxypeptidase B, nor does it affect the ability of pepsin to clot milk at pH 4.9.
DI S C U S S I O N
The heterogeneity of swine colostrum trypsin inhibitor observed previously 1 with the crystalline preparations was confirmed by disc electrophoresis. The question arose, do all bands observed in electrophoresis have the same activity, that is, are they isoinhibitors? The results presented in Fig. I show that the four substances can be called isoinhibitors, since they have essentially the same specific activity (within experimental error). Also, each fraction is homogeneous by disc electrophoresis (Figs. 3 and 4). Multiple forms of trypsin inhibitors have previously been noted in lima beans 18-21, soybeans 22, dog submandibular glands 23, and porcine pancreatic juice 24,25. In these cases the inhibitor forms differed by two or more amino acid residues. In the present report, only a single amino acid difference was detected among the four isoinhibitors (lack of lysine in Isoinhibitor II). The carbohydrate moiety appears to be responsible for the difference between Isoinhibitors IV and V, and no explanation is available for the separation of Isoinhibitors III and IV. The results shown in Table II suggest that the composition of "Fraction I" resembles that of the isoinhibitors except that in addition it contains an excess of some amino acids corresponding to a peptide Argl-Asp3-Ser3-Gluls-Pro13-Gly 8. The composition of this peptide is reminiscent of proteins of connective tissue. Incomplete synthesis is a possible explanation for the lack of a carboxyterminal lysine in Peak II. No preparative steps were employed that could conceivably have resulted in peptide bond hydrolysis. The carboxy-terminal sequence of the other isoinhibitors is most likely Thr-Lys-COOH. The presence of the carbohydrate moiety had been overlooked in the previous work 1. Its role is as yet unknown. Removal of sialic acid without loss of inhibitor activity has previously been noted with chicken ovomucoid 2a and with a-I inhibitor from human serum zv. This would be expected from the mechanism of inhibition ~8.
Biochim. Biophys. Acta, 229 (I97 x) 836-844
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L.F. KRESS et al.
ACKNOWLEDGMENTS
This study was supported by Grant AM-Io48I from the National Institute of Arthritis and Metabolic Diseases and Grant PRP-3o from the American Cancer Society. Portions of the data in this paper are taken from a thesis submitted by Miss Susan R. Martin to the Graduate School faculty, State University of New York at Buffalo, in partial fulfillment of the requirements for the degree of Master of Arts. The authors gratefully acknowledge the following for their assistance in providing colustrum: Dr. R. H. Grummer, University of Wisconsin; Mr. Stanley Slubecky, Erie County Penitentiary Farm, Alden, N.Y. ; and Dr. W. G. Pond, N.Y. State Experimental Station, Cornell University, Ithaca, N.Y. REFERENCES I M. LASKOWSKI, SR., B. KASSELL AND G. HAGERTY, Biochim. Biophys. Acta, 24 (1957) 300. 2 S. R. MARTIN, Master's Thesis, State University of New Y o r k at Buffalo, 1969. 3 M. LASKOWSKI, SR., in P. S. COLOWlCK AND N. O. KAPLAN, Methods of Enzymology, Vol. 2 Academic Press, New York, 1955, P- 16. 4 G. W. SCHWERT AND Y. TAKENAKA, Biochim. B~ophys. Aeta, 16 (1955) 57 °. 5 B. KASSELL, M. RADICEVIC, S. BERLOW, R. J. PEANASKY AND 1~{.LASKOWSKI, SR., J. Biol. Chem., 238 (1963) 3274 . 6 S. MOORE AND W. H. STEIN, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Voh 6, Acacemic Press, New York, 1964, p. 819. 7 C. H. W. HIRS, J. Bzol. Chem., 219 (1956) 611. 8 W. F. LENHARDT AND R. J. WINZLER, J. Chromatog., 34 (1968) 471. 9 J. T. CASSIDY, G . W . JOURDIAN AND S. ROSEMAN, in E. F. NEUEELD AND V. GINSBURG, Methods in Enzymology, Voh 8, Academic Press, New York, 1966, p. 680. IO G. L. ELLMANN, Arch. Biochem. Biophys., 82 (1959) 7 °. I I B. J. DAVIS, Ann. N . Y . Aead. Sci., 121 (1964) lO 4. 12 R. A. REISFELD, U. J. L E w i s AND D. E. WILLIAMS, Nature, 195 (1962) 281. 13 T. W. G o o n w l N AND R. A. MORTON, B*ochem. J., 4 ° (1946) 628. 14 E. SVENNERHOLM AND L. SVENNERHOLM, Nature, 181 (1958) 1154. 15 W. R. GRAY, in C. H. W. HIRS, Methods *n Enzymology, Voh I I , Academic Press, New York, 1967, p. 139. 16 G. R. STARK, ill C. H. W. HIRS, Methods *n Enzymology, Voh I I , Academic Press, New York, 1967 , p. 125. 17 A. GOTTSCHALK, Glycoprote,ns, B B A Library, Vol. 5, Elsevier, A m s t e r d a m , 1966. 18 G. JONES, S. MOORE AND W. H. STEIN, Biochemistry, 2 (1963) 66. 19 A. L. CARILLO, M.S. Thesis, M a r q u e t t e U n i v e r s i t y ,1962. 20 S. MATTHAI, M. S. Thesis, M a r q u e t t e University, 1963. 21 R. HAYNES AND R. E. FEENEY, J. Biol. Chem., 242 (1967) 5378. 22 V. FRATTALI AND R. V. STEINER, Biochemistry, 7 (1968) 521. 23 H. FRITZ, I. TRAUTSCHOLD, H. HAENDLE AND E. WERLE, Ann. N . Y . Acad. Sei., 146 (1968) 400. 24 L. J. GREENE, J. J. DICARLO, A. J. SUSSMAN AND D. C. BARTELT, J. B,ol. Chem., 243 (1968) 1804. 25 H. TSCHESCHE, E. WACHTER, S. I~UPFER AND K. NIEDERMEIER, Z. Physzol. Chem., 35 ° (1969) 1247. 26 R. E. FEENEY, M. B. RHODES AND J. S. ANDERSON, J. Biol. Chem., 235 (196o) 2633. 27 H. E. SCHULTZE, K. HEIDE AND H. HAUPT, Klin. Woehschr., 4 ° (1962) 427 • 28 M. LASKOWSKI, JR. AND R. W. SEALOCK, in P. D. BOYER, The Enzymes, Vol. 3, Academic Press, N e w York, 3rd ed., 1971, in the press.
Biochim. Biophys. Acta, 229 (1971) 836-844
BIOCHIMICA ET BIOPHYSICAACTA
BBA 35767 ISOLATION OF ISOINHIBITORS OF TRYPSIN FROM PORCINE COLOSTRUM
L A W R E N C E F. KRESS, SUSAN R. MARTIN* AND M. LASKOWSKI, SR.
Laboratory of Enzymology, Roswell Park Memomat Inst,tute, and the Department of Biochemzstry, Roswell Park Dwzsion, State Universzty of New York at Buffalo, Buffalo, N.Y. 14203 (U.S.A .) (Received September i4th, i97o)
SUMMARY
With the aid of chromatography on CM-cellulose the trypsin inhibitor from swine colostrum was subfractionated into four isoinhibitors. Each isoinhibitor migrated as a single band in disc electrophoresis at pH 4.3, and had the same specific activity against trypsin. All isoinhibitors contained carbohydrate. Only small differences in composition of isoinhibitors were detected. Sialic acid was present in one of them, and its removal did not alter inhibitor activity. The isoinhibitors are active against bovine and porcine trypsin, a-chymotrypsin and chymotrypsin B. They are inactive against carboxypeptidase B and the milk clotting activity of pepsin at pH 4.9. The reactive site of the inhibitor involves a lysine-X bond.
INTRODUCTION
Several years ago 1, a trypsin inhibitor was discovered in swine colostrum. It was isolated in a crystalline form, but showed some heterogeneity when subjected to electrophoresis. Recently, MARTINs devised a more efficient method of purification of the inhibitor. The preparation contained carbohydrate (II%) and was heterogeneous by disc electrophoresis. This preparation served as a starting material for the separation of individual isoinhibitors, using equilibrium chromatography and disc electrophoresis as criteria of purity. The purification, composition, and some properties of the isoinhibitors are now reported. MATERIALS AND METHODS
Bovine trypsin (twice crystallized), carboxypeptidases A and B (DFP treated), a-chymotrypsin and pepsin were obtained from Worthington. Porcine trypsin was from Novo Industry. Chymotrypsin B was prepared in this laboratory 3. BenzoylL-arginine ethyl ester hydrochloride was from Mann. Glucosamine and galactosamine Present address : Department of Biology, State University of New York at Buffalo.
BiochiA. Biophys. Acta, 229 (I97I) 836--844
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were from Pfanstiehl Laboratories, Inc. CM-cellulose was purchased from Schleicher and Schuell. Microgranular celluloses, CM-52 and DE-52, preswollen, were obtained from H. Reeve Angel and Co. Sephadex G-5 o was from Pharmacia. Porcine colostrum was collected for periods up to 36 h post partum. The samples were frozen as soon as possible and stored frozen until used.
Assays Trypsin and trypsin inhibitory activities were determined by the method of SCHWERT A N D TAKENAKA4, as modified by KASSELL et al. 5. Specific inhibitory activity was expressed as/zg of trypsin inhibited per I.O A2s0 nm unit of inhibitor*. Analytical methods Amino acid analyses were performed according to MOORE AND STEINs using a Beckman-Spinco I2oB analyzer. Approx. 3-mg samples of inhibitor were hydrolyzed in 8 ml of 5.7 M HC1 for 48 h at IIO °. Cystine was determined as cysteic acid 7. Amino sugars were determined b y hydrolyzing 2-mg samples in I.O ml of 4 M HC1 for 4 h at IOO°. Samples (0.2 ml) were applied directly to a 0. 9 cm × 15 cm column of the analyzer and eluted with p H 5.28 buffer. Neutral sugars were determined by gasliquid chromatography according to the method of LENHARDT AND WINZLER8. The authors are grateful to Dr. R. J. Winzler and to Mr. John Bleecher for the analysis. Sialic acid was determined by the thiobarbituric acid test described by CASSIDY et al. 9. Free sulthydryl was estimated according to ELLMANN10. Disc gel electrophoresis was performed by the method of DAVIS11 at p H 9.5 and according to REISFELD et al. 12at p H 4.3. The authors are indebted to Dr. Sally Schneider for these experiments. Carboxypeptidase determinations Approx. 3.o-mg samples of inhibitor dissolved in o.oi M sodium borate-o.i M NaC1 (pH 8.0) were incubated with 0.095 mg of carboxypeptidase B at 23 °. At suitable intervals a o.I-ml aliquot of the reaction was pipetted into 0. 9 ml of the p H 2.2 amino acid analyzer buffer and frozen until analysis. For carboxypeptidase A analysis, 4.9 mg of inhibitor (Peak II, Fig. 2) was dissolved in 0. 4 ml of 0.2 M LiC1. Carboxypeptidase A (o.13 mg) was added, the p H adjusted to 8.2, and the reaction allowed to proceed at 23 °. Aliquots (o.I ml) were withdrawn, pipetted into 0. 9 ml of p H 2.2 diluting buffer and frozen until analyzed. RESULTS
Purification of trypsin inhibitor Table I summarizes the results for a typical batch of colostrum. Step L Swine colostrum was thawed and centrifuged at 18 ooo x g for I.O h at o °. The fat layer at the top, and the residual pellet at the bottom were devoid of activity and were discarded. Step 2. To the liquid, an equal volume of 5 % trichloroacetic acid was added slowly, and stirring was continued for 2 h at room temperature. The precipitate was * P r o t e i n w a s e x p r e s s e d in A2sonm units. One u n i t refers t o t h e a m o u n t which, i f di s s ol ve d in i ml, w o u l d h a v e a n a b s o r b a n c e of u n i t y a t 28o rim.
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TABLE I SUMMARY OF PURIFICATION
PROCEDURE
Purification step
Total *nh*bitor (units X
R a w colostrum after centrifugation Trichloroacetic acid fractionation (NH4)=SO 4 fractionation CM-cellulose c h r o m a t o g r a p h y Sephadex G-5 o Gradient c h r o m a t o g r a p h y on microg r a n u l a r CM-cellulose
% Recovery Specific of inhibitory inhzb~tory act*vity activzty
10 6)
5.86 3.i8 3.7 ° 3.31 2.42
ioo 54.3 63.2 56.5 41.3
--
--
44oo 4815 575 ° 6o00
filtered on W h a t m a n No. 50 filter paper. The filter cake was re-extracted twice, with 2.5% trichloroacetic acid, and discarded. Step 3. Solid (NH4)2SO * was added to the combined filtrates to attain 80% saturation (603 g/l). After standing overnight at room temperature, the precipitate was collected on W h a t m a n No. 50 filter paper, dissolved in o.oi M ammonium formate (pH 3.7) and dialyzed against the same buffer at 2 °. The slightly turbid solution was clarified by centrifugation. Step 4. The inhibitor was charged on a 4 cm × 60 cm CM-52 cellulose column. The first peak was discarded. The starting buffer was replaced with buffer containing 0.3 M NaC1, A second peak contained 75 % of the inhibitory activity. It was pooled, dialyzed against distilled water, lyophilized, and stored at --18 °. Step 5. The active material was dissolved in o.oi M HCI-o. 3 M NaC1 (pH 2.1) and passed through a 5 cm × IOO cm column of Sephadex G-5o equilibrated with the same solution. The breakthrough peak contained no activity. Tubes which had a specific activity greater than 5500 units/A2s0 nm unit were pooled, concentrated, 0.350 0.300 t,t
0.2 z_
0
~0.Z00
O
tD +
0.100
ao
60
ioo
t4o
teo
TUBE No.
Fig. I. G r a d i e n t c h r o m a t o g r a p h y of swine colostrum t r y p s i n inhibitor on m i c r o g r a n u l a r CMcellulose. Swine eolostrum inhibitor (91 A2s0 nm units) was dissolved in 15 ml of o.oi M sodium succinate (pH 5.5) and dialyzed against the same buffer for 17 h. The solution was placed on a 1.5 cm × 88 cm column of m i c r o g r a n u l a r CM-cellulose. The protein was eluted w i t h a linear gradient f r o m o.oi succinate (i.o 1) to o.oi succinate-o.2 M NaC1 (i.o 1). Flow rate was 3 ° ml/h, and 5-ml fractions were collected. The average specific activity of Peak I was 3000 units/A 280 nm unit; t h a t of Peaks I I - V averaged 60o0 units/A=s8 nm u n i t in three separate c h r o m a t o g r a p h i c runs. , A 2 s 0 nm; O - - C ) , m o l a r i t y (suceinate + NaCI); [ [, t u b e s pooled.
B,och,m. Biophys. Acta, 229 (1971) 836-844
839
TRYPSIN INHIBITORS FROM PORCINE COLOSTRUM 0.350
0.350 -
0.300
0.300
~
oo200
0.200
i
,g
~t
0300
2O
40
60
80
~00
20
40
60
80
100
TUBE N O .
TUBE N o .
0.350 0.300
0.35c 0.30C
00.200 00.20C
0.J00 O.;OC
20 TUBE No.
40 60 80 TUBE N O .
100
Fig. 2. E q u i l i b r i u m c h r o m a t o g r a p h y of individual swine colostrum t r y p s i n inhibitors on microg r a n u l a r C1Vi-cellulose. I n all the above figures, the R o m a n n u m e r a l s correspond to those used in Fig. I. E a c h inhibitor was dissolved in IO ml of the respective buffer to be used and dialyzed against t h a t buffer. The protein solutions were placed on a 1,5 cm × 88 cm c o l u m n of microg r a n u l a r CM-cellulose and eluted w i t h the following buffers: Peak I I (22.8 A,80 nm units) : o.oi M s o d i u m succinate (pH 5.5); Peak I I I (35.9 -4,80 nm units): o.oi M succinate-o.oi 5 M NaC1; Peak I V (24.2 A,80 nm units): o.oi M succinate-o.o3 o M NaC1; Peak V (27. 5 A280 nmunits) : o.oi M succinate-o.o45 M NaC1. The flow rates were 3 ° ml/h, and 5-ml fractions were collected. The void v o l u m e of the columns occurred at T u b e io.
adjusted to p H 4.5 and dialyzed against distilled water. The material was lyophilized and stored at --18 °. Disc electrophoresis revealed at least six bands. Specific activity was 5750 units/A,80 nm unit. Step 6. The accumulated material (usually IOO A280 nm units) was dissolved in o.oi M sodium succinate (pH 5.5). The solution was dialyzed, charged on a column of CM-52, and eluted with a linear gradient of o.oi M sodium succinate (pH 5.5) and o.oi M sodium succinate-o.2 M NaC1 (pH 5.5). The results of a typical experiment are shown in Fig. I. The protein fractions were assigned Roman numerals. Fraction I consisted of heterogeneous material of low specific activity. Peaks II, I I I , IV, and V all had activity approaching 6000 units/A 2s0 nm unit. Recovery amounted to about 9 ° %. The peak tubes were pooled, concentrated, and stored at 4 ° . Material from five separate experiments was combined, dialyzed against water, and lyophilized. Step 7. Material from each peak obtained from the previous step was subjected to equilibrium chromatography on CM-52. NaC1 concentrations were so chosen that the inhibitor peak would emerge only after at least three void volumes had passed through the column. The column and the inhibitor solution were carefully equilibrated with the buffer used, prior to chromatography. The results are shown in Biochim. Biophys. Acta, 229 (I971) 836-844
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L.F. KRESS et al.
Fig. 3. P o l y a c r y l a m i d e gel electrophoresis of swine c o l o s t r u m t r y p s i n inhibitors. A p p r o x . 425 # g of t h e inhibitor fraction purified b y e q u i l i b r i u m c h r o m a t o g r a p h y were applied to a 1 5 % acryla m i d e s e p a r a t i n g gel a t p H 4.3. Electrophoresis was p e r f o r m e d at 5 m A per gel for 16o rain. T h e arrow m a r k s t h e position of t h e t r a c k i n g d y e ( b r o m p h e n o l blue) at t h e e n d of t h e run. T h e gels were s t a i n e d in 1 % aniline blue-black a n d d e - s t a i n e d b y leaching w i t h IO°//.o acetic acid. T h e R o m a n n u m e r a l s d e s i g n a t i n g t h e fractions correspond to t h o s e u s e d in Fig. I.
0.120
®
®
0.090 0~0.0£0 ....ii 0"5
0.030
20 40
IO
80I
I
I00
O
I
120 140 160 180 200
TUBE No. Fig. 4. Purification of P e a k I I I i n h i b i t o r o n CM-cellulose. P e a k I I I i n h i b i t o r (see Fig. 2) w a s dissolved in 5.5 ml o f o.oi M s o d i u m s u c c i n a t e - o . o i 5 M NaC1-6 M u r e a (pH 5.15) a n d placed on a 1.5 c m × 88 c m c o l u m n of m i c r o g r a n u l a r CM-cellulose e q u i l i b r a t e d w i t h t h e s a m e buffer. T h e initial h e t e r o g e n e o u s p e a k lacked i n h i b i t o r a c t i v i t y . T h e arrow i n d i c a t e s t h e p o i n t at w h i c h a linear g r a d i e n t of o.o15-o. 5 M NaC1 in o.oi M s u c c i n a t e - 6 M u r e a (pH 5.15) was initiated. T h e disc electrophoresis p a t t e r n of t h e m a t e r i a l w h i c h e m e r g e d is s h o w n a b o v e t h e peak. T h e inhib i t o r y m a t e r i a l h a d a specific a c t i v i t y of 585 ° units]A280 am unit. - - , A2s0 nm; , M NaC1.
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Fig. 2. All of the main peaks were well resolved and chromatographed symmetrically. The material from each peak was pooled, concentrated by rotary evaporation, dialyzed against distilled water, and lyophilized. This lyophilized material was used for analyses.
Disc electrophoresis The patterns from disc electrophoresis at pH 4-3 of the inhibitor peaks are shown in Fig. 3. All the inhibitor peaks gave single bands except Peak III which appeared as a double band. Peak I was heterogeneous as expected. Since Peak III exhibited two bands in both the pH 4.3 and pH 9.5 electrophoresis systems, it was subjected to further purification. Equilibrium chromatography of Peak III under the conditions used for Peaks II and IV showed that the material was not a mixture of Peaks II and IV. The successful purification of Peak III is illustrated in Fig. 4- Gradient chromatography at pH 5.1 in the presence of 6 M urea resulted in the removal of inactive material followed by the appearance of an active peak which migrated as a single band in disc electrophoresis. TABLE II AMINO ACID AND CARBOHYDRATE COMPOSITION OF INHIBITORS ISOLATED FROM SWINE COLOSTRUM Results are expressed as residues per molecule based on a value of isoleucine = i.oo.
Component
Peak I
Peak I I
Peak I I I
Peak I V
Peak V
Lysine Arginine Aspartic acid Threonine* Serine* Glutamic acid Proline Glycine Alanine Cystine** Valine Methionine*'* Isoleucine Leucine Tyrosine Phenylalanine
1.72 5.Ol 9.49 3.84 5.26 18.95 19.74 12.83 5 .°1 6 2.53 o.59 (i.oo) 4.3 ° 2.39 2.99
1.23 3.56 5.81 4.27 2.21 5.84 6.46 4.66 5.05 6.19 1.98 o.71 (i.oo) 4.09 2.44 3.76
2. io 3.72 6.08 4.31 2.22 6.13 6.79 4.85 5.18 6 2.2o 0.74 (i.oo) 4 .28 2.54 3.69
2.17 3.68 6.o9 4.25 2.14 5.88 6.71 4.80 5.15 6 2.38 o.81 (i.oo) 4 .18 2.50 3.61
1.97 3.8o 6.02 4.23 2.o8 5.66 6.92 4.91 5.29 6 2. i i o.82 (i.oo) 3.98 2.55 3.28
Glucosamine Galactosamine Sialic acid
1.94 o.32 o.61
3.7 ° o.39 0.80
3.74 o.46 o.oo
3.82 o.41 0.06
3.29 o.35 o.oi
Fucose Mannose Galactose Glucose
0.23 1.44 1.o6 o.o7
0.24 1.85 I.O6 o.04
0.38 2.22 1.41 Trace
0.42 1,79 1.42 o
0.38 2.o 7 i. 19 o.95
* N o t corrected for decomposition d u r i n g hydrolysis. ~* D e t e r m i n e d as cysteic acid in performic acid-oxidized Peak II. The values for the o t h e r peaks were a s s u m e d to be t h e same, since the recoveries of half-cystine were the s a m e for all peaks. "'* Uncorrected for decomposition; however, the value for methionine sulfone in performic acid-oxidized Peak I I was o.94 residue.
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L.F. KRESS et al.
Amino acid analyses The results of amino acid analysis are shown in Table II. The Roman numerals correspond to the peaks in Fig. I. Fraction I had a composition quite different from the other four. Cystine was determined on Peak I I as cysteic acid. The value for Peak I I was taken as the value for all the peaks, since the recovery of half-cystine in acid hydrolysates had been the same for all the peaks. All the values were corrected for 9% moisture. There were no differences in the amino acid composition of Peaks II, III, IV, and V, except for the lack of a lysine in Peak II. This was confirmed by the carboxypeptidase determinations (see below). Histidine and tryptophan (determined spectrophotometrically 13) were absent. Carbohydrate analysis Table I I shows that all peaks contained four glucosamine residues and only a trace of galactosamine. Only Isoinhibitor I I contained sialic acid, and no change in activity was noted after removing it with neuraminidase. All the sialic acid was shown to be the N-acetyl form in the butanol-propanol-HC1 chromatography system of SVENNERHOLM AND SVENNERHOLM14. The results of gas-liquid chromatography determinations of neutral sugars are also shown in Table II. The only significant difference among the active peaks is the presence of 0.95 residue of glucose in Peak V. C-terminal amino acid Incubation with carboxypeptidase B resulted in the rapid release of one residue of lysine from Isoinhibitors 11I, IV, and V. No significant amount of lysine was released from Isoinhibitor II, even after 6 h. On the other hand, exposure of Peak I I to carboxypeptidase A resulted in the slow release of threonine (o.53 residue in 21 h). No other amino acids were released by either of the carboxypeptidases in any of the inhibitor peaks. It was concluded that Isoinhibitors I l l to V all had a carboxyterminal lysine. Isoinhibitor I I differed from the others by one less lysine, and its C-terminal amino acid was threonine. N-terminal The dansyl chloride technique of GRAY15 and the cyanate procedure of STARK 16 were performed on both the intact and performic acid-oxidized inhibitor. All these methods failed to reveal the amino terminal residue, which is not unusual with glycoproteins 1~. Free sulfhydryl determination Treatment with the ELLMANN10 reagent indicated absence of free sulfnydryl groups, which led to the conclusion that the inhibitor possesses three disulfide bonds (Table II). Reactive site Colostrum inhibitor (3.0 mg of Peak I I material) was incubated with o.8 mg of porcine trypsin in 0.75 ml of o.ooi M HCI-o.o2 M CaC12 (pH 3.4) at 23 °. After 96 h the p H was adjusted to 8.0 with 0.2 M borate (pH 8.0) containing I.O M NaC1. Carboxypeptidase B (o.II mg in 30 #1) was added, and the reaction continued for 72 h. The p H was then adjusted to 2.o with 0.2 M HC1, and the reaction mixture Biochim. Biophys. ,4cta. 229 (1971) 836--844
TRYPSIN INHIBITORS FROM PORCINE COLOSTRUM
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frozen for amino acid analysis. The blank consisted of 0.8 mg of trypsin incubated at pH 3.4 for 9 6 h at which time the pH was adjusted to 8.0, and 3.0 mg of Peak II inhibitor and o.II mg of carboxypeptidase B were added. After 72 h the mixture was adjusted to pH 2.0. A net release of o.II #mole of lysine was observed in the experimental over the control tube while no arginine was detected. The amount of lysine recovered was 33% of that expected for full conversion to modified inhibitor. The actual net amount of lysine released from the inhibitor may be even higher, since the blank was set up to correct for the maximum possible release of lysine or arginine from tryptic autolysis products. It was concluded that the reactive site of the swine colostrum inhibitor involves a lysine-X bond.
Inhibitory activity with other proteolytic enzymes Swine colostrum inhibitor is also active against a-chymotrypsin, chymotrypsin B, and porcine trypsin. Removal of the sialic acid residue does not affect inhibitory activity in any of the above cases. The colostrum inhibitor does not inactivate carboxypeptidase B, nor does it affect the ability of pepsin to clot milk at pH 4.9.
DI S C U S S I O N
The heterogeneity of swine colostrum trypsin inhibitor observed previously 1 with the crystalline preparations was confirmed by disc electrophoresis. The question arose, do all bands observed in electrophoresis have the same activity, that is, are they isoinhibitors? The results presented in Fig. I show that the four substances can be called isoinhibitors, since they have essentially the same specific activity (within experimental error). Also, each fraction is homogeneous by disc electrophoresis (Figs. 3 and 4). Multiple forms of trypsin inhibitors have previously been noted in lima beans 18-21, soybeans 22, dog submandibular glands 23, and porcine pancreatic juice 24,25. In these cases the inhibitor forms differed by two or more amino acid residues. In the present report, only a single amino acid difference was detected among the four isoinhibitors (lack of lysine in Isoinhibitor II). The carbohydrate moiety appears to be responsible for the difference between Isoinhibitors IV and V, and no explanation is available for the separation of Isoinhibitors III and IV. The results shown in Table II suggest that the composition of "Fraction I" resembles that of the isoinhibitors except that in addition it contains an excess of some amino acids corresponding to a peptide Argl-Asp3-Ser3-Gluls-Pro13-Gly 8. The composition of this peptide is reminiscent of proteins of connective tissue. Incomplete synthesis is a possible explanation for the lack of a carboxyterminal lysine in Peak II. No preparative steps were employed that could conceivably have resulted in peptide bond hydrolysis. The carboxy-terminal sequence of the other isoinhibitors is most likely Thr-Lys-COOH. The presence of the carbohydrate moiety had been overlooked in the previous work 1. Its role is as yet unknown. Removal of sialic acid without loss of inhibitor activity has previously been noted with chicken ovomucoid 2a and with a-I inhibitor from human serum zv. This would be expected from the mechanism of inhibition ~8.
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ACKNOWLEDGMENTS
This study was supported by Grant AM-Io48I from the National Institute of Arthritis and Metabolic Diseases and Grant PRP-3o from the American Cancer Society. Portions of the data in this paper are taken from a thesis submitted by Miss Susan R. Martin to the Graduate School faculty, State University of New York at Buffalo, in partial fulfillment of the requirements for the degree of Master of Arts. The authors gratefully acknowledge the following for their assistance in providing colustrum: Dr. R. H. Grummer, University of Wisconsin; Mr. Stanley Slubecky, Erie County Penitentiary Farm, Alden, N.Y. ; and Dr. W. G. Pond, N.Y. State Experimental Station, Cornell University, Ithaca, N.Y. REFERENCES I M. LASKOWSKI, SR., B. KASSELL AND G. HAGERTY, Biochim. Biophys. Acta, 24 (1957) 300. 2 S. R. MARTIN, Master's Thesis, State University of New Y o r k at Buffalo, 1969. 3 M. LASKOWSKI, SR., in P. S. COLOWlCK AND N. O. KAPLAN, Methods of Enzymology, Vol. 2 Academic Press, New York, 1955, P- 16. 4 G. W. SCHWERT AND Y. TAKENAKA, Biochim. B~ophys. Aeta, 16 (1955) 57 °. 5 B. KASSELL, M. RADICEVIC, S. BERLOW, R. J. PEANASKY AND 1~{.LASKOWSKI, SR., J. Biol. Chem., 238 (1963) 3274 . 6 S. MOORE AND W. H. STEIN, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Voh 6, Acacemic Press, New York, 1964, p. 819. 7 C. H. W. HIRS, J. Bzol. Chem., 219 (1956) 611. 8 W. F. LENHARDT AND R. J. WINZLER, J. Chromatog., 34 (1968) 471. 9 J. T. CASSIDY, G . W . JOURDIAN AND S. ROSEMAN, in E. F. NEUEELD AND V. GINSBURG, Methods in Enzymology, Voh 8, Academic Press, New York, 1966, p. 680. IO G. L. ELLMANN, Arch. Biochem. Biophys., 82 (1959) 7 °. I I B. J. DAVIS, Ann. N . Y . Aead. Sci., 121 (1964) lO 4. 12 R. A. REISFELD, U. J. L E w i s AND D. E. WILLIAMS, Nature, 195 (1962) 281. 13 T. W. G o o n w l N AND R. A. MORTON, B*ochem. J., 4 ° (1946) 628. 14 E. SVENNERHOLM AND L. SVENNERHOLM, Nature, 181 (1958) 1154. 15 W. R. GRAY, in C. H. W. HIRS, Methods *n Enzymology, Voh I I , Academic Press, New York, 1967, p. 139. 16 G. R. STARK, ill C. H. W. HIRS, Methods *n Enzymology, Voh I I , Academic Press, New York, 1967 , p. 125. 17 A. GOTTSCHALK, Glycoprote,ns, B B A Library, Vol. 5, Elsevier, A m s t e r d a m , 1966. 18 G. JONES, S. MOORE AND W. H. STEIN, Biochemistry, 2 (1963) 66. 19 A. L. CARILLO, M.S. Thesis, M a r q u e t t e U n i v e r s i t y ,1962. 20 S. MATTHAI, M. S. Thesis, M a r q u e t t e University, 1963. 21 R. HAYNES AND R. E. FEENEY, J. Biol. Chem., 242 (1967) 5378. 22 V. FRATTALI AND R. V. STEINER, Biochemistry, 7 (1968) 521. 23 H. FRITZ, I. TRAUTSCHOLD, H. HAENDLE AND E. WERLE, Ann. N . Y . Acad. Sei., 146 (1968) 400. 24 L. J. GREENE, J. J. DICARLO, A. J. SUSSMAN AND D. C. BARTELT, J. B,ol. Chem., 243 (1968) 1804. 25 H. TSCHESCHE, E. WACHTER, S. I~UPFER AND K. NIEDERMEIER, Z. Physzol. Chem., 35 ° (1969) 1247. 26 R. E. FEENEY, M. B. RHODES AND J. S. ANDERSON, J. Biol. Chem., 235 (196o) 2633. 27 H. E. SCHULTZE, K. HEIDE AND H. HAUPT, Klin. Woehschr., 4 ° (1962) 427 • 28 M. LASKOWSKI, JR. AND R. W. SEALOCK, in P. D. BOYER, The Enzymes, Vol. 3, Academic Press, N e w York, 3rd ed., 1971, in the press.
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