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Isolation and Partial Characterization of a Native Serine-Type Protease Inhibitor from Bovine Milk
Isolation and Partial Characterization of a Native Serine-Type Protease Inhibitor from Bovine Milk B. A. WEBER' and S. S. NIELSEN2 Department of Food Science Purdue University West Lafayetle, IN 47907 ABSTRACT
Purification of a native serine-type protease inhibitor from raw bovine milk resulted in the isolation of an inhibitor tentatively identified as al-antitrypsin. Techniques utilized included ammonium sulfate fractionation followed by metal chelate, hydrophobic interaction, and gel filtration chromatography. Inhibitory activity during the isolation procedure was monitored using benzoyl-DL-arginine-pnitroanilide. Molecular weight range of the isolated inhibitor (56,000 to 64,OOO kDa) was comparable with that of human serum al-antitrypsin (60,000ma). The pH optima were 6.0 and 7.5. The isolated inhibitor was active against trypsin and elastase, but activity against plasmin was not detectable. The inhibitor was a glycoprotein and formed an SDS staple complex with elastase. (Key words: milk, milk protease inhibitor, serine protease inhibitor)
Abbreviation key: ACA = aminocaproic acid, al-AT = a l - a n t i t ~ ~ ~ saz-AP in, = e-antiphmin, a2-M = upmacroglobulin, BAPNA = benzoyl-DL-arginine-p-nitroanilide,dd = deionized distilled, DMSO = dimethyl sulfox,, = K average, TEMED = tetraide, K methylethylene diamine. INTRODUCTION
Proteolytic activity in normal bovine milk has been attributed both to naturalIy occurring proteases and to proteases produced by contam-
Received M a y 17, 1990. Accepted October 22, 1990. 'Present address: Kraft General Foods, Inc., Battle Creek, MI 49017. 2I'o whom correspondence should be addressed. 1991 J Dairy Sci 74:764-771
hating bacteria (15, 23). Because of their partial resistance to heat, native proteases can contribute to the proteolysis and gelation observed in sterilized milk products (1, 10, 32). These proteases also release bitter peptides that influence the flavor of cheeses and UHT-processed milk (14, 15). Native milk proteases as well as their activators and inhibitors must be considered when studying the stability of milk and milk products. Plasmin, the major protease naturally present in milk, exists primarily in its inactive form, plasminogen (31). F'lasminogen must be converted to plasmin by other proteases @e., plasminogen activators) present in milk before degradation of milk proteins by plasmin can occur (33). Native milk protease inhibitors reportedly act on plasminogen activators and plasmin (31, 34). Although little information is available on native protease inhibitors in bovine milk, extensive literature is available on inhibitors found in human blood. The major protease inhibitors in human plasma are al-antitrypsin (al-AT), qmacroglobulin (a+), and crpantiplasmin (012AP) (3). Molecular size of %-M precludes its diffusion from blood to milk (3). However, the smaller molecular sizes of al-AT and q - A P would allow them to be native protease inhibitors in normal bovine milk. Lindberg (25) concluded that the protease inhibitor activity in human is mainly a function of al-AT. The inhibitors al-AT and q - M differ from the major bovine colostral inhibitor in that they are larger in molecular weight and are not acid and heat stable (3, 21, 22). The inhibitor al-AT is a single chain glycoprotein with a molecular weight of approximately 60,OOO kDa, a carbohydrate content of 11.5 to 16%, and no disulfide bridges. The inhibitor %-AP is also a single chain glycoprotein with a molecular weight of approximately 65,000 to 70,000kDa, a carbohydrate content of 14%, and three disulfide
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SERINE-TYPE PROTEASE INHIBITOR
bridges. Human blood al-AT irreversibly inhibits trypsin, chymotrypsin, elastase, and plasmin as well as several other serum proteins in a 1:l enzyme to inhibitor ratio (2, 3). Plasmin is inhibited very rapidly by CQ-AP,whereas CQAP inhibits trypsin and other trypsin-like enzymes slowly (21). Isolation of al-AT from human blood has been achieved using ammonium sulfate fractionation, metal chelate, and ion exchange chromatography (24, 36). Due to the milder conditions employed, yield and homogeneity of al-AT isolated by this procedure are much better than when Concanavalin A or blue dextran is used to remove albumin. The high lysine affinity of a2-AP has been utilized during its isolation from human blood (24). Normal bovine milk exhibits trypsin inhibitor activity as determined by gel or plate diffusion methods (5, 6, 33, but the inhibitor has yet to be isolated. Most attempts to isolate protease inhibitors from bovine milk have included an acid treatment to extract interfering proteins such as casein (34).These attempts have not been successful, presumably because the acid treatment inactivated the inhibitor. Using milder isolation procedures, a native milk protease inhibitor was isolated and then partially characterized in terms of quantity, pH optima for activity, inhibition pattern, and molecular weight. MATERIALS AND METHODS Collection and Preparation of Whey
Raw whole milk obtained from the Purdue University Dairy Center bulk storage tank was skimmed by centrifugation (2800 x g for 20 min at 4'C) in a Beckman model J2-21 (Beckman Instruments, Inc., Fullerton, CA) refrigerated centrifuge. Following adjustment to .05 M 6-aminocaproic acid (ACA) (Aldrich Chemical Co., Milwaukee, WI) and stining at room temperature for 1 h, the skim milk was centrifuged in a Beckman Preparative Ultracentrifuge model L3-50 using a SW-28 rotor at 135,000x g for 1 h at 4'C. This procedure effectively precipitated casein from the whey. Whey samples prepared during several ultracentrifugation runs were pooled for immediate use in the isolation procedure.
Determination of tnhlbltor Activity
Inhibitor activity was estimated by measuring the decrease in activity of trypsin on the
synthetic substrate benzoy1-DL-arginine-pnimanilide (BAPNA) (Sigma Chemical Co., St. Louis, MO). The method of Kakade et al. (19) was used, with modifications as described The BAPNA (30 mg) was dissolved in 1 ml of dimethyl sulfoxide (DMSO) before dilution to 100 ml with .05 M Tris-HC1 (PH 8.2, .02 M CaCl2). Trypsin (EC 3.4.21.4, type m),5 mg in 100 ml .001 M HC1, was used. Samples were diluted 150 with Tris-HC1 (pH 8.2, .02 M CaC12). To a .5 ml aliquot of diluted sample and .5 ml of trypsin solution, 2 ml of BAPNA solution were added. Reaction time at 37 "C was exactly 10 min, after which .5 ml of 30% acetic acid was added. An appropriate sample blank was prepared for each sample. Absorbance at 410 nm was determined using a Gilford Response spectrophotometer (Ciba Corning Diagnostics Corp., Medfield, MA). Percentage of inhibition was calculated as follows: A410 trypsin
-
(A410 sample - A410 sample blank) A410 trypsin
x 100 = percentage of inhibition. Protein Determination
Protein content of samples and fractions was estimated by the Lowry procedure using bovine serum albumin in deionized distilled (dd) water as the standard (27). Chromatographic Methods
Protein elution for all chromatographic methods was monitored by absorbance at 280 nm with a Perkin-Elmer model 571 spectrophotometer (Perkin-Elmer, Norwalk, Cr). Fractions from protein peaks were tested for inhibitory activity using BAPNA. Metal Chelate Chromatography. Sepharose 6-B (Pharmacia Fine Chemicals, Piscataway, NJ) was activated and coupled with ZnCl2 according to Porath et al. (30). The column (2 x 40 cm) was equilibrated with .1 M sodium phosphate (pH 8.0, .15 M NaC1). Flow rate was 50 ml/h, and 10-ml fractions were collected. Hydrophobic Interaction Chromatography. Phenyl-Sepharose (Pharmacia) was prepared Joumal of Dairy Science Vol. 74, No. 3, 1991
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according to manufacturer's directions and poured into a 1 x 10 cm column. Equilibration Made to 50% (NH4)2SO4 buffer was .1 M sodium phosphate (pH 7.5,2.5 Rtcipitate M ammonium sulfate). Flow rate was 40 muh, discarded <1 and 2 ml fractions were collected. A gradient of Supematant made to 2.5 to 0 M ammonium sulfate in .1 M sodium 80% (NH4)2so4 phosphate buffer @H 7.0) was used to elute sup-t J discardad
The isolation procedure is summarized in Figure 1. Whole milk was treated and whey was prepared as described previously. To 500 ml of whey, 500 ml of saturated ammonium sulfate solution were added to yield a solution of 50% saturated with ammonium sulfate. Mixture was stirred at room temperature for 1 h before stirring overnight at 4'C. After cenaifugation at 15,000 x g for 20 min at 4°C. the supernatant was decanted and placed in an ice water bath. During reextraction, precipitate was resuspended in 250 ml of 50% saturated ammonium sulfate and stirred at room temperature for 1 h before centrifugation. To the pooled supernatants. ammonium sulfate (232.8 g) was added to achieve 80% saturation. After stirring overnight at 4'C and centrifugation at 15 ,OOO x g for 20 min at 4'C. resultant Supernatant was discarded. Precipitate was resolubilized in .1 M sodium phosphate buffer @H 8.0, .15 M NaCl) and dialyzed 3 d against the same buffer. Dialyzed sample was applied to the Zn chelate column. Column was washed with dialyzing buffer until absorbance at 280 nm was <.loo. Protein with inhibitory activity was eluted by changing to a .05 M sodium phosphate buffer @H 6.5, .15 M NaCl). Fractions with inhibitor activity were pooled, adjusted to 2.5 M ammonium sulfate, and applied to the phenylSepharose column. Fractions that contained protein peaks eluted with the ammonium sulfate gradient were dialyzed overnight against .1 M sodium phosphate buffer @H 7.0) prior to determining inhibitory activity with BAF'NA. Fractions with activity were pooled and applied Journal of Dairy Science Vol. 74, No. 3, 1991
1 1
Made to 2.5 M (NH4)2SO4 Applied to phe~I~l-Sepharo~e CO~WILU
J.
Eluted with gradient of 2.5 to .O M (N?I4)2SO4
J.
Pooled fractions from phenyl-Sepharose column
L
Dialyzed against .1 M sodium phosphate buffer, p H 7.5
L
Applied to Sephadex G-100 column
J.
Pooled fractions from Sephade~G-100 column
J.
Lyophilized
Figure 1. Summary of procedure to isolate protease inbibitor from bovine whey.
to the Sephadex G-100 size exclusion column. Eluted fractions with inhibitory activity were pooled and lyophilized. Specific activity after each purification step was calculated by dividing percentage of inhibition by milligrams of protein in the pooled fractions. Characterization
Molecular Weight Determination. Molecular weight of isolated inhibitor was estimated by its K average ( K,) from a Sephadex G-100 column prepared as previously described. Molecular weights O a ) of proteins used to standardize the column were bovine serum albumin, 66,ooO, chicken egg albumin, 45,000;
SERINE-TYPE PROTEASE INHIBITOR
carbonic anhydrase, 29,000; cytochrome C, 12,400; and aprotinin, 6500 (Sigma). Blue dextran (1 mglml) was used to determine the void volume of the column. Total column volume was determined by using dinitrophenyl-glycine. The K, values of the standard proteins were calculated as follows: K, = [mention volume (V,) - void volume (Vo)]/[total column volume (VJ - void volume (VO)].The Kav values were plotted against their respective log molecular weights, and the molecular weight of the isolated inhibitor was estimated. pH Optimum Determination. Citrate phosphate, sodium phosphate, and Tris buffers at .1 M were prepared in .5 pH unit interials covering pH ranges 5.0 to 6.0, 6.0 to 7.5, and 7.5 to 9.5, respectively. purified inhibitor from the size exclusion chromatography step was resolubilized in 2 ml of dd water. Activity assay utilized BAPNA as described with the following exceptions: a) after dissolving BAPNA in DMSO, an aliquot of this solution was diluted with each pH buffer being tested and b) sample was diluted in each pH buffer being tested rather than in the pH 8.2 Tris buffer. Electrophoretic Techniques. The SDSPAGE was performed by the method of Fling and Gregerson (12). The 10% gels were silver stained according to the method of Merril et al. (28). Inhibitor and glycoprotein stained gels were done according to the methods of Davis (9), as modified by Niekemp et al. (29) and Fnels (13) and Dubray and B e d (11). respectively. Isoelectric focusing was conducted according to LKB Application Note 250 (26) with the following changes: acry1amide:bisacrylamide percentage was increased to lo%, glycerol content was decreased to 4 ml, and 60 pl of N,N,”,”-tetra-methylethylene diamine (TEMED) was added to promote polymerization. Broad range pH gradient of pH 3.5 to 9.5 was prepared by combining appropriate ampholines (LKB, Bromma, Sweden) as indicated in the application note (26). Specificity. Inhibitory activity against trypsin was determined using BAPNA by the method of Kakade et al. (19) as described above. Inhibition of elastase was determined using the method of Bieth et al. (4) with N-succinyl-(Lalanyl)3-p-nitroanilide (Sigma) as substrate. Inhibitory activity against plasmin was tested using the method of Korycka-Dahl et al. (20) with the substrate Val-leu-lys-p-nitroanilide(S-2251) (Sigma).
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Figure 2. chromatogramfrom Zn chelate chromatography of bovine whey; (--) absorbance at 280 mn, (a-0) specific activity. Specific activity was determined using percentage inhibition from the benzoyl-arginine-pnimanilide assay divided by milligmms of protein.
RESULTS AND DISCUSSION Isolation
The procedure used by Johnson and Travis (17, 18) to isolate al-AT from human serum required modification, as noted, for use with bovine milk. The initial ammonium sulfate fractionation step was as in the isolation of alAT from blood (17, 18). Resolubilized precipitate from ammonium sulfate fractionation was applied to the Zn chelate chromatographic (Figure 2) column. Inherent low level of inhibitor in milk and high concentration of other whey proteins apparently made the Zn chelateinhibitor interaction difficult and thereby resulted in a low amount of activity being eluted from the Zn chelate column. To remove interfering whey proteins, tandem copper and zinc-chelate columns were tested as described by Porath (30). Tandem columns were not effective, in that all activity was found in one fraction from the Cu chelate column wash (data not shown). Therefore, the Z n chelate column alone was retained as the second step in the isolation procedure. To isolate inhibitor from blood, ion exchange chromatography on a DE-52 column at pH 7.5 was the third step in the isolation procedure. However, with bovine milk, ion exchange chromatography on DE-52 at pH conditions of 6.5, 7.0, 7.5, and 8.0 as well as on CM-52 at pH 7.0 yielded no protein peak with a matching peak in activity (data not shown). Consequently, separation based on differences in Journal of Dairy Science Vol. 74, No. 3, 1991
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WEBER AND
weight of 64,OOO m a . Staining procedures revealed that the protein contained carbohydrate and was an active inhibitor (not shown). The slight shift to an apparent lower molecular weight as isolation progressed likely was due to partial loss of the glycosidic portion of the inhibitor. Table 1 summarizes the activity and purification from each step in the isolation procedure. Starting whey (500 ml) had a total protein content of 5610 mg. Due to cloudiness of the whey, inhibitor activity was not easily determined and therefore not reported. Specific acFigure 3. Chromatogram from phenyl-Sepharose chromatography of Zn chelate fractions (50 to 70); (----) absor- tivity during purification increased from 28 bance at 280 nm, (0-0) specific activity. Specific activity (percentage of inhibition per milligram of prowas determined using percentage inhibition from the ben- tein) for the 50% ammonium sulfate supemazoyl-arghiue-pnitroanilideassay divided by milligrams of tant to 6600 for the final pooled fractions from protein. the Sephadex G-100 column. The small amount of material recovered is in agreement with other reports (5, 34). hydrophobicity of proteins was tested. Use of a phenyl-Sepharose column resulted in elution of Characterization a single large protein peak with activity peaks Molecular Weight Determination. Molecular on either side (Figure 3). No other fractions contained inhibitory activity. Fractions 33 to 36 weight estimates of the inhibitor as determined and 40 to 45 were pooled and subjected to size by size exclusion chromatography and SDSexclusion chromatography using Sephadex G- PAGE were 56,700 and 64,000 kDa, respec100 fine (Figure 4). The major activity peak tively. This range is well within that (41,000 to was associated with the second protein peak. Each step in the isolation procedure was monitored by SDS-PAGE (Figure 5). The purification techniques resulted in the isolation of a single protein with an apparent molecular F R A C T I O N *"I.CR
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Figure 4. Chromatogram from Sephadex G-100 chromatography of phenylSepharose fractions (33 to 36,40 to 45); (----)absorbance at 280 mn, (0-0) specific activity. Specific activity was determined using percentage inhibition from the benzoyl-arginine-pnitmanilideassay divided by miuigrams of protein. Journal of Dairy Science Vol. 74, No. 3, 1991
n
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e
a m
nb ~
c
d
e
i
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h
Figure 5. The SDS-polyacrylamide gel of samples during procedure to isolate protease inbibitor from bovine mille a) molecular weight standards, b) whey, c) 50% (NHQ)2SO4 supernatant,d) 80% cNH4)2SO4 precipitate, e)
Zn chelate pooled fraction, f) phenyl-Sepharose pooled fraction, g) Sqhadex G-100pooled fraction^ (60to 75). h) molecular weight standards.
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SERINE-TYPE PROTEASE INHIBITOR TABLE 1. Summary of purification of protease inhibitor from bovine whey.
Purification step
specific activity'
Whey 50% (NHq)2SO4 supernatant 80% (NHq)zSO4 precipitate Zn chelate pooled fractions Phenyl-Sepbitrose pooled fractions Sephade~G-100 poolad ~ I W ~ ~ O U S
ND4 28 38 118 1600
6600
Total protein (mg)
Units
activityz
Mcation factor3
ND
ND
total
5610 2860 1032 9.0 .5 .15
1
80,080 39.216 1.062 800
1.4 42 57.1 235.7
990
'Specific activity = Percentage inhibition of trypsin divided by milligrams of protein. 2Activity measured by benzoyl-arginine-pnitroaoilideassay at pH 8.2 and calculated by multiplying specific activity by total protein. 'Purification factor determined using specific activity starting with the 50% (NH4)2SO4 Supernatant. 'hl= Not determined.
60,000 kDa) reported for a l - A T isolated from human and bovine blood (3). The glycosidic nature of the inhibitor (11.5 to 16%) likely contributed to the wide range in molecular weight estimates, because percentage of carbohydrate moiety can be altered depending on the severity of isolation conditions used (3). Specificity. Specificity of the inhibitor was determined against trypsin, elastase, and plasmin. Specific activity against trypsin was 6600 (Table 1), whereas against elastase, it was 13,600 (percentage of inhibition per milligram of protein). An SDS gel showed that the inhibitor isolated from bovine milk formed an SDSstable complex with elastase (gel not shown). Attempts under various conditions failed to demonstrate inhibition of plasmin. Perhaps a p propriate conditions for activity against plasmin were not used. As was the case for human blood al-AT (3), future studies may demonstrate plasmin inhibition by this inhibitor isolated from bovine mik. High inhibitory activity obtained toward trypsin and elastase is consistent with the specificity reported for a l - A T , namely, elastase, followed by trypsin. plasmin, and thrombin (7). pH Optimum. Optimum activity of the isolated inhibitor was obtained at pH 6.0 and 7.5, although activity was also detected at pH 8.5 and 9.5 (Figure 6). This dual major pH optimum was somewhat unexpected and may explain the ineffectiveness of ion exchange chromatography in the isolation scheme. Because trypsin controls were run at each pH, effect of pH on trypsin activity was not the cause of the
dual pH optimum. Perhaps charge differences at or near the active site of the inhibitor resulted in increased activity at more than one pH. Also, isoinhibitors might exist. Existence of a l - A T isoinhibitors is well documented. Beminger (3) in a review article on al-AT cited much of the literature concluding that variability in a l - A T was due to slight genetic variances. Isoelectric point values reported ranged from pH 5.10 f .05 with 6 bands (8) to pH 4.5 to 4.7 with 4 to 6 bands (16). An isoelectric focusing gel of the inhibitor isolated from bovine milk also exhibited several bands (gel not shown). Heterogeneity was most pronounced when pooled starting material was used (5). Whey used in the present isolation
I
50
6
' 3
I
10
8
I3
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Figure 6. pH optimum determination of inhibitor is* lated from bovine milk. Inhibitory activity measured using the benzoyl-arginine-pnitxoanilide assay; o citrate phosphate buffer, 0 sodium phosphate buffer, A Tris buffer. Journal of Dairy Science Vol. 74, No. 3. 1991
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was from bulk tank milk and represented milk pooled from several cows. Consequently, the dual pH optimum observed for the isolated inhibitor appears reasonable. CONCLUSIONS
The inhibitor isolated from bovine milk using ammonium sulfate fractionation followed by Zn chelate, hydrophobic interaction, and size exclusion chromatography compared favorably in terms of molecular weight and specificity with al-AT isolated from various mammalian sources by using similar techniques. Staining procedures used with polyacrylamide gels indicated that the isolated inhibitor is a glycoprotein that forms a SDSstable complex with elastase, as is true for human serum al-AT. Consequently, this inhibitor from bovine milk has been identified tentatively as al-AT. The putative al-AT isolated from bovine milk is to our knowledge the only native protease inhibitor that has been purified to homogeneity. Although experiments to date suggest that this protease inhibitor does not inhibit plasmin, larger amounts of the inhibitor must be isolated to conduct further characterization experiments. This inhibitor and other native milk protease inhibitors, particularly az-AP, must be studied further to determine their importance in controlling the proteolysis of milk proteins. ACKNOWLEDGMENTS
This work was supported in part through a grant provided by the National Dairy Promotion and Research Board. This paper is Number 12481 of the Purdue University Agricultural Experiment Station. REFERENCES
H.Wrathhall. and A. T. Andrews. 1986. Heat stability of plasnin (milk proteinase) and plasminogen. J. Dairy Res. 53259. 2Beatty, K., J. Bieth, and J. Travis. 1980. Kinetics of association of serine proteinases with native and oxidized a-1-proteinase inhibitor and a-l-antichymotrypsin. J. Biol. Chem. 255:3531. 3Beminger, R. W. 1985. Alphal-antitrypsin. J. Med. (Westbury) 16:23. 4Bieth. J., B. Spiess, and C. G. Wcnnuth. 1974. The synthesis and analytical use of a highly sensitive and convenient substrate of elastase. Biochem. Med. 11: 350. 1Alichanidis, E., J.
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5Buzalski-Homhen, T., T. Kat& and M. Sandholm. 1981. Milk and antitrypsin activity during clinical and -tal bovine mastitis. Acta Vet. Scaud.22360. 6 B u z a l s k i - H ~ ~T.,k and ~ ~ M. Sandholm. 1981. try^ sin-inhibitor in mastitic milk and colostrum: correlation between trypsin-inhibitor capacity, bovine s e m albumin and somatic cell counts. J. Dairy Res. 48:213. 7 Carrell, R. W.,J . 4 . Jeppssop, C.-B. Laurell, S. 0. Brenoaq U C. Owen, L. Vaughn, and D. R Boswell. 1982. Stracture and variation of human al-antitrypsin. Nature (Lond.) 298329. 8Crawford. I. P. 1973. Ruification and properties of normal human alpha-1-antitrypsin. Arch. Biochem. Biophys. 156:215. 9Davis, B. J. 1964. Disc electrophoresis. II. Methods and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404. lODriesscn, F. M., and C. B. van der Waals. 1978. Inactivation of native milk proteinase by heat treatment. Neth. Milk Dairy J. 32245. 11 Dubray, G., and G. Bezard. 1982. A h i o y sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels. Anal. Biochem. 119:325. 12Fling, S. P.,and D. S. Gregerson. 1986. Peptide and protein molecular weight determination by electrophoresis using a high-molarity triS buffer system without urea. Anal. Biochem. 155:83. 13Friels, J. M. 1986. pnrification and characterhtion of 2 trypsin inhibitors from prosomillet (Panicum milliocewn). U S . Thesis, Univ. Nebrash-Lincoln. 14Harwalker, V. R 1982. Age gelation of sterilized millrs. Page 229 in Developments in dairy chemistry. 1. Proteins. P.F. Fox,ed. Awl. Sci. Publ.. London, Engl. 15Humbert, G., and C. Alais. 1979. Review of the progress of dairy science: the milk proteinase system. J. Dairy Res. 46559. 16 Jeppsson, J.-O., C.-B. Laurell, and M. Fagerhol. 1978. Properties of isolated human alph~-l-autitrypsinsof Pi types M , S and Z. Eur. J. Biochem. 83:143. 17Johnson, D., and J. Travis. 1978. Structural evidence for methionine at the reactive site of human ci1-proteinase inhibitor. J. Biol. Chem. 2537142. 18 Johnson, D., and I. Travis. 1979. The oxidative inactivation of human a-1-proteinase inhibitor. J. Biol. Chcm. 2544022. 19 Kakade, M. L., N. Simons, and I. E. Lienex. 1969. An evaluation of n a h d vs. synthetic substrates for measuxing the antibyptic activity of soybean samples. Cereal Chem. 46518. 20Korycka-Dahl, M., B. R Dumas, N. Chene, and J. Martal. 1983. plasmin activity in millr. J. Dairy Sci. 6 6 704. 21Laskowski, M.,Jr., and K. Ikunoskia 1980. Protein inhibitors of proteinases. Annu. Rev. Biochem. 49593. 22 Laskowski, M., Jr., and M.Laskowski. 1951. Crystalline trypsin inhibitor fium colostnun. J. Biol. Chm. 190563. 23Law, B. A. 1979. Reviews of the progress of dairy science: enzymes of psychrohophic bacteria and their effects on miIk and milk products. J. Dairy Res. 46: 573. 24Lijen, H. R, and D. Collen. 1985. Alpha-2-antiplasmin. J. Med. (Westbury) 16225. 25 Lindberg, T. 1979. Protease inhibitors in human milk. Pediatr. Res. 13:969.
SERINE-TYPE PROTEASE INHIBITOR 26 LKB. 1977. Analytical electrofocusing in thin layers of polyacrylamide gels. LKB Application Note 250. LKB, Bromma, Sweden. 27 Lowry, 0. H.,N. J. Rosebrough, A. L. Parr. and R J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265. 28 Merril, C. R, D. Goldman, S. A. Sedmau, aud M. H. E M . 1981. Ultnlsensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211:1437. 29Niekamp. C. A., H.I. HiLson, and M. LasLowski, Jr. 1%9. Peptide-bond hydrolysis equilibria in native proteins. Conversion of virgin into modified soybean trypsin inhibitor. Biochemistry 8:16. 30Porath, J., and B. Oh. 1983. ImmobiJjzed metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials, serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 221621. 31 Richardson, €3. C. 1983. Variation of the concentration
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of plasminogen in bovine milk with lactation. N.Z. J. Dairy Sci. Techuol. 18247. 32 Snoeren, T.H.M., C. A. van der Spek, R. Delsker, and P. Both. 1979. Proteolysis during the storage of UHTsterilized whole milk. 1. Experiments with milk heated by the direct system for 4 seconds at 142'C. Neth. Mik Dairy J. 3331. 33 Violand, B. N., and €7. J. Castellino. 1976. Mechanism of urokinase-catalyzed activation of human plasminogen. I. Biol. Chem. 251:3906. 34von Emst, H.,E.H.Reimerdes, H. Klostermeyer, and E. Sayk. 1976. Milk proteases 7. Fractionation of components of the proteinase inhibitor system in milk. Milchwissenschaft 31:325. 35von Fellenkg, R., and H. Horber. 1980. Multiple proteinase inhibitors in colostrum, prepartal rymmary secretion, milk and mammary tissue. Schweu. Arch. Tiaheilkd. 122:159. 36 Wiman, €3. 1981. Human c~-antiplasmin.Methods Enzymol. 80:395.
Journal of Dairy Science Vol. 74. No. 3, 1991
Purification of a native serine-type protease inhibitor from raw bovine milk resulted in the isolation of an inhibitor tentatively identified as al-antitrypsin. Techniques utilized included ammonium sulfate fractionation followed by metal chelate, hydrophobic interaction, and gel filtration chromatography. Inhibitory activity during the isolation procedure was monitored using benzoyl-DL-arginine-pnitroanilide. Molecular weight range of the isolated inhibitor (56,000 to 64,OOO kDa) was comparable with that of human serum al-antitrypsin (60,000ma). The pH optima were 6.0 and 7.5. The isolated inhibitor was active against trypsin and elastase, but activity against plasmin was not detectable. The inhibitor was a glycoprotein and formed an SDS staple complex with elastase. (Key words: milk, milk protease inhibitor, serine protease inhibitor)
Abbreviation key: ACA = aminocaproic acid, al-AT = a l - a n t i t ~ ~ ~ saz-AP in, = e-antiphmin, a2-M = upmacroglobulin, BAPNA = benzoyl-DL-arginine-p-nitroanilide,dd = deionized distilled, DMSO = dimethyl sulfox,, = K average, TEMED = tetraide, K methylethylene diamine. INTRODUCTION
Proteolytic activity in normal bovine milk has been attributed both to naturalIy occurring proteases and to proteases produced by contam-
Received M a y 17, 1990. Accepted October 22, 1990. 'Present address: Kraft General Foods, Inc., Battle Creek, MI 49017. 2I'o whom correspondence should be addressed. 1991 J Dairy Sci 74:764-771
hating bacteria (15, 23). Because of their partial resistance to heat, native proteases can contribute to the proteolysis and gelation observed in sterilized milk products (1, 10, 32). These proteases also release bitter peptides that influence the flavor of cheeses and UHT-processed milk (14, 15). Native milk proteases as well as their activators and inhibitors must be considered when studying the stability of milk and milk products. Plasmin, the major protease naturally present in milk, exists primarily in its inactive form, plasminogen (31). F'lasminogen must be converted to plasmin by other proteases @e., plasminogen activators) present in milk before degradation of milk proteins by plasmin can occur (33). Native milk protease inhibitors reportedly act on plasminogen activators and plasmin (31, 34). Although little information is available on native protease inhibitors in bovine milk, extensive literature is available on inhibitors found in human blood. The major protease inhibitors in human plasma are al-antitrypsin (al-AT), qmacroglobulin (a+), and crpantiplasmin (012AP) (3). Molecular size of %-M precludes its diffusion from blood to milk (3). However, the smaller molecular sizes of al-AT and q - A P would allow them to be native protease inhibitors in normal bovine milk. Lindberg (25) concluded that the protease inhibitor activity in human is mainly a function of al-AT. The inhibitors al-AT and q - M differ from the major bovine colostral inhibitor in that they are larger in molecular weight and are not acid and heat stable (3, 21, 22). The inhibitor al-AT is a single chain glycoprotein with a molecular weight of approximately 60,OOO kDa, a carbohydrate content of 11.5 to 16%, and no disulfide bridges. The inhibitor %-AP is also a single chain glycoprotein with a molecular weight of approximately 65,000 to 70,000kDa, a carbohydrate content of 14%, and three disulfide
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765
SERINE-TYPE PROTEASE INHIBITOR
bridges. Human blood al-AT irreversibly inhibits trypsin, chymotrypsin, elastase, and plasmin as well as several other serum proteins in a 1:l enzyme to inhibitor ratio (2, 3). Plasmin is inhibited very rapidly by CQ-AP,whereas CQAP inhibits trypsin and other trypsin-like enzymes slowly (21). Isolation of al-AT from human blood has been achieved using ammonium sulfate fractionation, metal chelate, and ion exchange chromatography (24, 36). Due to the milder conditions employed, yield and homogeneity of al-AT isolated by this procedure are much better than when Concanavalin A or blue dextran is used to remove albumin. The high lysine affinity of a2-AP has been utilized during its isolation from human blood (24). Normal bovine milk exhibits trypsin inhibitor activity as determined by gel or plate diffusion methods (5, 6, 33, but the inhibitor has yet to be isolated. Most attempts to isolate protease inhibitors from bovine milk have included an acid treatment to extract interfering proteins such as casein (34).These attempts have not been successful, presumably because the acid treatment inactivated the inhibitor. Using milder isolation procedures, a native milk protease inhibitor was isolated and then partially characterized in terms of quantity, pH optima for activity, inhibition pattern, and molecular weight. MATERIALS AND METHODS Collection and Preparation of Whey
Raw whole milk obtained from the Purdue University Dairy Center bulk storage tank was skimmed by centrifugation (2800 x g for 20 min at 4'C) in a Beckman model J2-21 (Beckman Instruments, Inc., Fullerton, CA) refrigerated centrifuge. Following adjustment to .05 M 6-aminocaproic acid (ACA) (Aldrich Chemical Co., Milwaukee, WI) and stining at room temperature for 1 h, the skim milk was centrifuged in a Beckman Preparative Ultracentrifuge model L3-50 using a SW-28 rotor at 135,000x g for 1 h at 4'C. This procedure effectively precipitated casein from the whey. Whey samples prepared during several ultracentrifugation runs were pooled for immediate use in the isolation procedure.
Determination of tnhlbltor Activity
Inhibitor activity was estimated by measuring the decrease in activity of trypsin on the
synthetic substrate benzoy1-DL-arginine-pnimanilide (BAPNA) (Sigma Chemical Co., St. Louis, MO). The method of Kakade et al. (19) was used, with modifications as described The BAPNA (30 mg) was dissolved in 1 ml of dimethyl sulfoxide (DMSO) before dilution to 100 ml with .05 M Tris-HC1 (PH 8.2, .02 M CaCl2). Trypsin (EC 3.4.21.4, type m),5 mg in 100 ml .001 M HC1, was used. Samples were diluted 150 with Tris-HC1 (pH 8.2, .02 M CaC12). To a .5 ml aliquot of diluted sample and .5 ml of trypsin solution, 2 ml of BAPNA solution were added. Reaction time at 37 "C was exactly 10 min, after which .5 ml of 30% acetic acid was added. An appropriate sample blank was prepared for each sample. Absorbance at 410 nm was determined using a Gilford Response spectrophotometer (Ciba Corning Diagnostics Corp., Medfield, MA). Percentage of inhibition was calculated as follows: A410 trypsin
-
(A410 sample - A410 sample blank) A410 trypsin
x 100 = percentage of inhibition. Protein Determination
Protein content of samples and fractions was estimated by the Lowry procedure using bovine serum albumin in deionized distilled (dd) water as the standard (27). Chromatographic Methods
Protein elution for all chromatographic methods was monitored by absorbance at 280 nm with a Perkin-Elmer model 571 spectrophotometer (Perkin-Elmer, Norwalk, Cr). Fractions from protein peaks were tested for inhibitory activity using BAPNA. Metal Chelate Chromatography. Sepharose 6-B (Pharmacia Fine Chemicals, Piscataway, NJ) was activated and coupled with ZnCl2 according to Porath et al. (30). The column (2 x 40 cm) was equilibrated with .1 M sodium phosphate (pH 8.0, .15 M NaC1). Flow rate was 50 ml/h, and 10-ml fractions were collected. Hydrophobic Interaction Chromatography. Phenyl-Sepharose (Pharmacia) was prepared Joumal of Dairy Science Vol. 74, No. 3, 1991
766
WEBER AND NIELSEN
according to manufacturer's directions and poured into a 1 x 10 cm column. Equilibration Made to 50% (NH4)2SO4 buffer was .1 M sodium phosphate (pH 7.5,2.5 Rtcipitate M ammonium sulfate). Flow rate was 40 muh, discarded <1 and 2 ml fractions were collected. A gradient of Supematant made to 2.5 to 0 M ammonium sulfate in .1 M sodium 80% (NH4)2so4 phosphate buffer @H 7.0) was used to elute sup-t J discardad
The isolation procedure is summarized in Figure 1. Whole milk was treated and whey was prepared as described previously. To 500 ml of whey, 500 ml of saturated ammonium sulfate solution were added to yield a solution of 50% saturated with ammonium sulfate. Mixture was stirred at room temperature for 1 h before stirring overnight at 4'C. After cenaifugation at 15,000 x g for 20 min at 4°C. the supernatant was decanted and placed in an ice water bath. During reextraction, precipitate was resuspended in 250 ml of 50% saturated ammonium sulfate and stirred at room temperature for 1 h before centrifugation. To the pooled supernatants. ammonium sulfate (232.8 g) was added to achieve 80% saturation. After stirring overnight at 4'C and centrifugation at 15 ,OOO x g for 20 min at 4'C. resultant Supernatant was discarded. Precipitate was resolubilized in .1 M sodium phosphate buffer @H 8.0, .15 M NaCl) and dialyzed 3 d against the same buffer. Dialyzed sample was applied to the Zn chelate column. Column was washed with dialyzing buffer until absorbance at 280 nm was <.loo. Protein with inhibitory activity was eluted by changing to a .05 M sodium phosphate buffer @H 6.5, .15 M NaCl). Fractions with inhibitor activity were pooled, adjusted to 2.5 M ammonium sulfate, and applied to the phenylSepharose column. Fractions that contained protein peaks eluted with the ammonium sulfate gradient were dialyzed overnight against .1 M sodium phosphate buffer @H 7.0) prior to determining inhibitory activity with BAF'NA. Fractions with activity were pooled and applied Journal of Dairy Science Vol. 74, No. 3, 1991
1 1
Made to 2.5 M (NH4)2SO4 Applied to phe~I~l-Sepharo~e CO~WILU
J.
Eluted with gradient of 2.5 to .O M (N?I4)2SO4
J.
Pooled fractions from phenyl-Sepharose column
L
Dialyzed against .1 M sodium phosphate buffer, p H 7.5
L
Applied to Sephadex G-100 column
J.
Pooled fractions from Sephade~G-100 column
J.
Lyophilized
Figure 1. Summary of procedure to isolate protease inbibitor from bovine whey.
to the Sephadex G-100 size exclusion column. Eluted fractions with inhibitory activity were pooled and lyophilized. Specific activity after each purification step was calculated by dividing percentage of inhibition by milligrams of protein in the pooled fractions. Characterization
Molecular Weight Determination. Molecular weight of isolated inhibitor was estimated by its K average ( K,) from a Sephadex G-100 column prepared as previously described. Molecular weights O a ) of proteins used to standardize the column were bovine serum albumin, 66,ooO, chicken egg albumin, 45,000;
SERINE-TYPE PROTEASE INHIBITOR
carbonic anhydrase, 29,000; cytochrome C, 12,400; and aprotinin, 6500 (Sigma). Blue dextran (1 mglml) was used to determine the void volume of the column. Total column volume was determined by using dinitrophenyl-glycine. The K, values of the standard proteins were calculated as follows: K, = [mention volume (V,) - void volume (Vo)]/[total column volume (VJ - void volume (VO)].The Kav values were plotted against their respective log molecular weights, and the molecular weight of the isolated inhibitor was estimated. pH Optimum Determination. Citrate phosphate, sodium phosphate, and Tris buffers at .1 M were prepared in .5 pH unit interials covering pH ranges 5.0 to 6.0, 6.0 to 7.5, and 7.5 to 9.5, respectively. purified inhibitor from the size exclusion chromatography step was resolubilized in 2 ml of dd water. Activity assay utilized BAPNA as described with the following exceptions: a) after dissolving BAPNA in DMSO, an aliquot of this solution was diluted with each pH buffer being tested and b) sample was diluted in each pH buffer being tested rather than in the pH 8.2 Tris buffer. Electrophoretic Techniques. The SDSPAGE was performed by the method of Fling and Gregerson (12). The 10% gels were silver stained according to the method of Merril et al. (28). Inhibitor and glycoprotein stained gels were done according to the methods of Davis (9), as modified by Niekemp et al. (29) and Fnels (13) and Dubray and B e d (11). respectively. Isoelectric focusing was conducted according to LKB Application Note 250 (26) with the following changes: acry1amide:bisacrylamide percentage was increased to lo%, glycerol content was decreased to 4 ml, and 60 pl of N,N,”,”-tetra-methylethylene diamine (TEMED) was added to promote polymerization. Broad range pH gradient of pH 3.5 to 9.5 was prepared by combining appropriate ampholines (LKB, Bromma, Sweden) as indicated in the application note (26). Specificity. Inhibitory activity against trypsin was determined using BAPNA by the method of Kakade et al. (19) as described above. Inhibition of elastase was determined using the method of Bieth et al. (4) with N-succinyl-(Lalanyl)3-p-nitroanilide (Sigma) as substrate. Inhibitory activity against plasmin was tested using the method of Korycka-Dahl et al. (20) with the substrate Val-leu-lys-p-nitroanilide(S-2251) (Sigma).
767
Figure 2. chromatogramfrom Zn chelate chromatography of bovine whey; (--) absorbance at 280 mn, (a-0) specific activity. Specific activity was determined using percentage inhibition from the benzoyl-arginine-pnimanilide assay divided by milligmms of protein.
RESULTS AND DISCUSSION Isolation
The procedure used by Johnson and Travis (17, 18) to isolate al-AT from human serum required modification, as noted, for use with bovine milk. The initial ammonium sulfate fractionation step was as in the isolation of alAT from blood (17, 18). Resolubilized precipitate from ammonium sulfate fractionation was applied to the Zn chelate chromatographic (Figure 2) column. Inherent low level of inhibitor in milk and high concentration of other whey proteins apparently made the Zn chelateinhibitor interaction difficult and thereby resulted in a low amount of activity being eluted from the Zn chelate column. To remove interfering whey proteins, tandem copper and zinc-chelate columns were tested as described by Porath (30). Tandem columns were not effective, in that all activity was found in one fraction from the Cu chelate column wash (data not shown). Therefore, the Z n chelate column alone was retained as the second step in the isolation procedure. To isolate inhibitor from blood, ion exchange chromatography on a DE-52 column at pH 7.5 was the third step in the isolation procedure. However, with bovine milk, ion exchange chromatography on DE-52 at pH conditions of 6.5, 7.0, 7.5, and 8.0 as well as on CM-52 at pH 7.0 yielded no protein peak with a matching peak in activity (data not shown). Consequently, separation based on differences in Journal of Dairy Science Vol. 74, No. 3, 1991
768
WEBER AND
weight of 64,OOO m a . Staining procedures revealed that the protein contained carbohydrate and was an active inhibitor (not shown). The slight shift to an apparent lower molecular weight as isolation progressed likely was due to partial loss of the glycosidic portion of the inhibitor. Table 1 summarizes the activity and purification from each step in the isolation procedure. Starting whey (500 ml) had a total protein content of 5610 mg. Due to cloudiness of the whey, inhibitor activity was not easily determined and therefore not reported. Specific acFigure 3. Chromatogram from phenyl-Sepharose chromatography of Zn chelate fractions (50 to 70); (----) absor- tivity during purification increased from 28 bance at 280 nm, (0-0) specific activity. Specific activity (percentage of inhibition per milligram of prowas determined using percentage inhibition from the ben- tein) for the 50% ammonium sulfate supemazoyl-arghiue-pnitroanilideassay divided by milligrams of tant to 6600 for the final pooled fractions from protein. the Sephadex G-100 column. The small amount of material recovered is in agreement with other reports (5, 34). hydrophobicity of proteins was tested. Use of a phenyl-Sepharose column resulted in elution of Characterization a single large protein peak with activity peaks Molecular Weight Determination. Molecular on either side (Figure 3). No other fractions contained inhibitory activity. Fractions 33 to 36 weight estimates of the inhibitor as determined and 40 to 45 were pooled and subjected to size by size exclusion chromatography and SDSexclusion chromatography using Sephadex G- PAGE were 56,700 and 64,000 kDa, respec100 fine (Figure 4). The major activity peak tively. This range is well within that (41,000 to was associated with the second protein peak. Each step in the isolation procedure was monitored by SDS-PAGE (Figure 5). The purification techniques resulted in the isolation of a single protein with an apparent molecular F R A C T I O N *"I.CR
I
L
~
o
~
~
m
~
s
m
m
w
~
~
FRACTION NUMeKW
Figure 4. Chromatogram from Sephadex G-100 chromatography of phenylSepharose fractions (33 to 36,40 to 45); (----)absorbance at 280 mn, (0-0) specific activity. Specific activity was determined using percentage inhibition from the benzoyl-arginine-pnitmanilideassay divided by miuigrams of protein. Journal of Dairy Science Vol. 74, No. 3, 1991
n
~
e
a m
nb ~
c
d
e
i
g
h
Figure 5. The SDS-polyacrylamide gel of samples during procedure to isolate protease inbibitor from bovine mille a) molecular weight standards, b) whey, c) 50% (NHQ)2SO4 supernatant,d) 80% cNH4)2SO4 precipitate, e)
Zn chelate pooled fraction, f) phenyl-Sepharose pooled fraction, g) Sqhadex G-100pooled fraction^ (60to 75). h) molecular weight standards.
769
SERINE-TYPE PROTEASE INHIBITOR TABLE 1. Summary of purification of protease inhibitor from bovine whey.
Purification step
specific activity'
Whey 50% (NHq)2SO4 supernatant 80% (NHq)zSO4 precipitate Zn chelate pooled fractions Phenyl-Sepbitrose pooled fractions Sephade~G-100 poolad ~ I W ~ ~ O U S
ND4 28 38 118 1600
6600
Total protein (mg)
Units
activityz
Mcation factor3
ND
ND
total
5610 2860 1032 9.0 .5 .15
1
80,080 39.216 1.062 800
1.4 42 57.1 235.7
990
'Specific activity = Percentage inhibition of trypsin divided by milligrams of protein. 2Activity measured by benzoyl-arginine-pnitroaoilideassay at pH 8.2 and calculated by multiplying specific activity by total protein. 'Purification factor determined using specific activity starting with the 50% (NH4)2SO4 Supernatant. 'hl= Not determined.
60,000 kDa) reported for a l - A T isolated from human and bovine blood (3). The glycosidic nature of the inhibitor (11.5 to 16%) likely contributed to the wide range in molecular weight estimates, because percentage of carbohydrate moiety can be altered depending on the severity of isolation conditions used (3). Specificity. Specificity of the inhibitor was determined against trypsin, elastase, and plasmin. Specific activity against trypsin was 6600 (Table 1), whereas against elastase, it was 13,600 (percentage of inhibition per milligram of protein). An SDS gel showed that the inhibitor isolated from bovine milk formed an SDSstable complex with elastase (gel not shown). Attempts under various conditions failed to demonstrate inhibition of plasmin. Perhaps a p propriate conditions for activity against plasmin were not used. As was the case for human blood al-AT (3), future studies may demonstrate plasmin inhibition by this inhibitor isolated from bovine mik. High inhibitory activity obtained toward trypsin and elastase is consistent with the specificity reported for a l - A T , namely, elastase, followed by trypsin. plasmin, and thrombin (7). pH Optimum. Optimum activity of the isolated inhibitor was obtained at pH 6.0 and 7.5, although activity was also detected at pH 8.5 and 9.5 (Figure 6). This dual major pH optimum was somewhat unexpected and may explain the ineffectiveness of ion exchange chromatography in the isolation scheme. Because trypsin controls were run at each pH, effect of pH on trypsin activity was not the cause of the
dual pH optimum. Perhaps charge differences at or near the active site of the inhibitor resulted in increased activity at more than one pH. Also, isoinhibitors might exist. Existence of a l - A T isoinhibitors is well documented. Beminger (3) in a review article on al-AT cited much of the literature concluding that variability in a l - A T was due to slight genetic variances. Isoelectric point values reported ranged from pH 5.10 f .05 with 6 bands (8) to pH 4.5 to 4.7 with 4 to 6 bands (16). An isoelectric focusing gel of the inhibitor isolated from bovine milk also exhibited several bands (gel not shown). Heterogeneity was most pronounced when pooled starting material was used (5). Whey used in the present isolation
I
50
6
' 3
I
10
8
I3
D
.I
PH
Figure 6. pH optimum determination of inhibitor is* lated from bovine milk. Inhibitory activity measured using the benzoyl-arginine-pnitxoanilide assay; o citrate phosphate buffer, 0 sodium phosphate buffer, A Tris buffer. Journal of Dairy Science Vol. 74, No. 3. 1991
770
WEBER AND "
was from bulk tank milk and represented milk pooled from several cows. Consequently, the dual pH optimum observed for the isolated inhibitor appears reasonable. CONCLUSIONS
The inhibitor isolated from bovine milk using ammonium sulfate fractionation followed by Zn chelate, hydrophobic interaction, and size exclusion chromatography compared favorably in terms of molecular weight and specificity with al-AT isolated from various mammalian sources by using similar techniques. Staining procedures used with polyacrylamide gels indicated that the isolated inhibitor is a glycoprotein that forms a SDSstable complex with elastase, as is true for human serum al-AT. Consequently, this inhibitor from bovine milk has been identified tentatively as al-AT. The putative al-AT isolated from bovine milk is to our knowledge the only native protease inhibitor that has been purified to homogeneity. Although experiments to date suggest that this protease inhibitor does not inhibit plasmin, larger amounts of the inhibitor must be isolated to conduct further characterization experiments. This inhibitor and other native milk protease inhibitors, particularly az-AP, must be studied further to determine their importance in controlling the proteolysis of milk proteins. ACKNOWLEDGMENTS
This work was supported in part through a grant provided by the National Dairy Promotion and Research Board. This paper is Number 12481 of the Purdue University Agricultural Experiment Station. REFERENCES
H.Wrathhall. and A. T. Andrews. 1986. Heat stability of plasnin (milk proteinase) and plasminogen. J. Dairy Res. 53259. 2Beatty, K., J. Bieth, and J. Travis. 1980. Kinetics of association of serine proteinases with native and oxidized a-1-proteinase inhibitor and a-l-antichymotrypsin. J. Biol. Chem. 255:3531. 3Beminger, R. W. 1985. Alphal-antitrypsin. J. Med. (Westbury) 16:23. 4Bieth. J., B. Spiess, and C. G. Wcnnuth. 1974. The synthesis and analytical use of a highly sensitive and convenient substrate of elastase. Biochem. Med. 11: 350. 1Alichanidis, E., J.
Journal of Dairy Science Vol. 74, No. 3, 1991
5Buzalski-Homhen, T., T. Kat& and M. Sandholm. 1981. Milk and antitrypsin activity during clinical and -tal bovine mastitis. Acta Vet. Scaud.22360. 6 B u z a l s k i - H ~ ~T.,k and ~ ~ M. Sandholm. 1981. try^ sin-inhibitor in mastitic milk and colostrum: correlation between trypsin-inhibitor capacity, bovine s e m albumin and somatic cell counts. J. Dairy Res. 48:213. 7 Carrell, R. W.,J . 4 . Jeppssop, C.-B. Laurell, S. 0. Brenoaq U C. Owen, L. Vaughn, and D. R Boswell. 1982. Stracture and variation of human al-antitrypsin. Nature (Lond.) 298329. 8Crawford. I. P. 1973. Ruification and properties of normal human alpha-1-antitrypsin. Arch. Biochem. Biophys. 156:215. 9Davis, B. J. 1964. Disc electrophoresis. II. Methods and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404. lODriesscn, F. M., and C. B. van der Waals. 1978. Inactivation of native milk proteinase by heat treatment. Neth. Milk Dairy J. 32245. 11 Dubray, G., and G. Bezard. 1982. A h i o y sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels. Anal. Biochem. 119:325. 12Fling, S. P.,and D. S. Gregerson. 1986. Peptide and protein molecular weight determination by electrophoresis using a high-molarity triS buffer system without urea. Anal. Biochem. 155:83. 13Friels, J. M. 1986. pnrification and characterhtion of 2 trypsin inhibitors from prosomillet (Panicum milliocewn). U S . Thesis, Univ. Nebrash-Lincoln. 14Harwalker, V. R 1982. Age gelation of sterilized millrs. Page 229 in Developments in dairy chemistry. 1. Proteins. P.F. Fox,ed. Awl. Sci. Publ.. London, Engl. 15Humbert, G., and C. Alais. 1979. Review of the progress of dairy science: the milk proteinase system. J. Dairy Res. 46559. 16 Jeppsson, J.-O., C.-B. Laurell, and M. Fagerhol. 1978. Properties of isolated human alph~-l-autitrypsinsof Pi types M , S and Z. Eur. J. Biochem. 83:143. 17Johnson, D., and J. Travis. 1978. Structural evidence for methionine at the reactive site of human ci1-proteinase inhibitor. J. Biol. Chem. 2537142. 18 Johnson, D., and I. Travis. 1979. The oxidative inactivation of human a-1-proteinase inhibitor. J. Biol. Chcm. 2544022. 19 Kakade, M. L., N. Simons, and I. E. Lienex. 1969. An evaluation of n a h d vs. synthetic substrates for measuxing the antibyptic activity of soybean samples. Cereal Chem. 46518. 20Korycka-Dahl, M., B. R Dumas, N. Chene, and J. Martal. 1983. plasmin activity in millr. J. Dairy Sci. 6 6 704. 21Laskowski, M.,Jr., and K. Ikunoskia 1980. Protein inhibitors of proteinases. Annu. Rev. Biochem. 49593. 22 Laskowski, M., Jr., and M.Laskowski. 1951. Crystalline trypsin inhibitor fium colostnun. J. Biol. Chm. 190563. 23Law, B. A. 1979. Reviews of the progress of dairy science: enzymes of psychrohophic bacteria and their effects on miIk and milk products. J. Dairy Res. 46: 573. 24Lijen, H. R, and D. Collen. 1985. Alpha-2-antiplasmin. J. Med. (Westbury) 16225. 25 Lindberg, T. 1979. Protease inhibitors in human milk. Pediatr. Res. 13:969.
SERINE-TYPE PROTEASE INHIBITOR 26 LKB. 1977. Analytical electrofocusing in thin layers of polyacrylamide gels. LKB Application Note 250. LKB, Bromma, Sweden. 27 Lowry, 0. H.,N. J. Rosebrough, A. L. Parr. and R J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265. 28 Merril, C. R, D. Goldman, S. A. Sedmau, aud M. H. E M . 1981. Ultnlsensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211:1437. 29Niekamp. C. A., H.I. HiLson, and M. LasLowski, Jr. 1%9. Peptide-bond hydrolysis equilibria in native proteins. Conversion of virgin into modified soybean trypsin inhibitor. Biochemistry 8:16. 30Porath, J., and B. Oh. 1983. ImmobiJjzed metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials, serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 221621. 31 Richardson, €3. C. 1983. Variation of the concentration
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of plasminogen in bovine milk with lactation. N.Z. J. Dairy Sci. Techuol. 18247. 32 Snoeren, T.H.M., C. A. van der Spek, R. Delsker, and P. Both. 1979. Proteolysis during the storage of UHTsterilized whole milk. 1. Experiments with milk heated by the direct system for 4 seconds at 142'C. Neth. Mik Dairy J. 3331. 33 Violand, B. N., and €7. J. Castellino. 1976. Mechanism of urokinase-catalyzed activation of human plasminogen. I. Biol. Chem. 251:3906. 34von Emst, H.,E.H.Reimerdes, H. Klostermeyer, and E. Sayk. 1976. Milk proteases 7. Fractionation of components of the proteinase inhibitor system in milk. Milchwissenschaft 31:325. 35von Fellenkg, R., and H. Horber. 1980. Multiple proteinase inhibitors in colostrum, prepartal rymmary secretion, milk and mammary tissue. Schweu. Arch. Tiaheilkd. 122:159. 36 Wiman, €3. 1981. Human c~-antiplasmin.Methods Enzymol. 80:395.
Journal of Dairy Science Vol. 74. No. 3, 1991