Selected Physico-Chemical Characteristics of Bovine Colostrum Immunoglobulin1

Selected Physico-Chemicol Characteristics of Bovine Colostrum Immunoglobulin' Abstract

Fractions rich in IgM were pooled and rechromatographed. Effluents with IgM activity were dialyzed against cold distilled water, precipitated with 50~ saturated (NH4)SO4, washed twice with cold distilled water, and resuspended in .15M NaC1. Polyacrylamide disc gel electrophoresis. For polyacrylamide disc gel electrophoresis at alkaline pH, the procedure of Davis (4) was used with Tris-glyeine buffer, pH 8.9, and gel strength of 7.5%. At the acid pH, acetic acidfi-alanine buffer, pH 4,5 and 7.5% gel strength, was used as suggested by Reisfeld et al. (14). Sedimentation constants. A Beckman Model E analytical ultracentrifuge equipped with Schlieren optics determined sedimentation constants. All experiments were carried out at a speed of 40,000 rpm (116,272 x g) in a pH 8.0, .1M Tris-HCl buffer containing .15M NaC1 at 20 C. Peak mobilities were measured with a Nikon Profile Projector Model 6C. 1% Extinction Coefficient (EZS0nm)"Protein coneentration of solutions of IgM in .1M TrisHC1 buffer (pH 8.0) was determined by micro-Kjeldahl method. Based upon nitrogen content of 14.5% reported for human IgM (11), a conversion factor of 6.90 was used. Absorbance of protein solution (Fig. 1) after appropriate dilution was measured at 280 nm by a Hitachi Perkin-Elmer Model 139 UVVIS spectrophotometer. Carbohydrate content. Hexose content of

Three immunoglobulin preparations isolated from bovine colostrum by ammonium sulfate ffactionation and repeated ion-exchange column chromatography on DEAE-Sephadex A-50 had sedimentation values ranging from 18.2 to 19.8 and extinction coefficients of 11.8 +__ .2. Carbohydrate content was 10.78 _+ .31% consisting of 5.13 ___ .12% hexoses, 1.25 _-+- .05% fucose, 1.40 ___ .05% sialic acid, and 2.90 ___ .09% hexosamines. The immunoglobulin preparations contained no free sulfhydryl groups. However, the total number of groups was 43.6 per mole of immunoglobulin based on an approximate molecular weight of 900,000, indicating that irnmunoglobulin contained 21 disulfide bonds per mole. Introduction

We have described isolation of individual immunoglobulin classes from bovine milk and colostrum (9). This paper reports on selected physico-chemical characteristics of the bovine IgM preparation. Methods and Materials

Bovine eolostrum obtained from cows within 24 hr after pa~urition was treated as described (10). Protein solution obtained after exhaustive dialys/s against .02M phosphate buffer, pit 8.0, was subjected to ion-exchange chromatography on a DEAE-Sephadex A-50 (1.5 x 30 cm) column equilibrated with the same buffer. Eluting buffers were .02M phosphate, pH 8.0, followed by .3M phosphate developing an ion gradient according to procedure of Heimer et al. (8). Effluent fractions were examined immunoelectrophoretieally (10) with anti-bovine serum, anti-bovine gamma globulin serum, and monospecifie anti-IgM serum obtained from commereial sourees; Dr. J. E. Butler, USDA, Washington, D.C.; and our own laboratory. Received for publication September 15, 19"/2. XApproved as Journal Series Article 86-72 of the Ohio Agricultural Research and Development Center, Columbus. This investigation was supported in part by Public Health Services Grant FD-0016I from the Office of Research and Training Grants, Food and Drag Administration.

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5.1 6.8 % PROTEIN

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Fie. 1. Effect of IgM concentration on UV absorption at 280 nm.

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IgM was determined by procedure of Miller and Metzger (11), sialic acid by thiobarbituric acid method of Warren (15) using bovine serum albumin as the blank, fucose by procedure of Dische and Shettles (5), and hexosamines according to Boas (2) using glucosamine-HC1 (Nutritional Biochem. Corp., Cleveland, Ohio) as the standard. Sulfhydryl groups and disulfide bonds. Reduction of disulfide bonds and estimation of resulting sulfhydryl (-SH) groups were made by procedure of Miller and Metzger (12). Selected conditions were reduction of 1~; solution of IgM in .2M Tris-HCI buffer, pH 8.6, with .001M dithiothreitol (Nutritional Biochemicals Corp., Cleveland, Ohio) at 25 C for 60 min and estimation of released -SH groups by Ellman's method (6) with 5M guanidine as the solvent and 5,5" Dithiobis (2-nitrogenzoic acid) reagent. fl-lactoglobulin was the standard. Results and Discussion

The IgM preparations showed a single peak upon analytical ultra-centrifugation, a single arc upon immunoelectrophoresis against selected anti-sera, and a single protein band at the spacer-sample gel interface at alkaline or acid pH electrophoresis (Fig. 2). Sedimentation constant ($2o) values of IgM were determined in .1M Tris-HC1 buffer, pH 8.0, containing .15M NaC1. The S2o.w values for three different concentrations of IgM were cal-

FIG. 2. P@acrylarnide disc gel electrophoretic (A), Schlieren (B), and immunoelectrophoretic (C) patterns of an IgM preparation. Polyaerylamide gel electrophoresis run in pH 8.9 (left) and pH 4.5 (right) buffer; Sehlieren pattern for 50 rain after IOURNAL OF DAIRY SCIENCE VOL.

56, NO.

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FIc. 3. Concentration dependence of sedimentation of IgM at pH 8.6 in .1M Tris-HC1 buffer containing .15M NaC1. culated and plotted (Fig. 3) to determine concentration dependence of sedimentation of IgM. When the S2o.w values for concentrations were extrapolated to infinite dilution, a S°20,w value of 19.3 was obtained. The $2o values from the other preparations of IgM from colostrum of four different cows ranged from 18.2 to 19.8. These values are within the range reported for human serum IgM (11) and by Mukkur and Froese (13) for bovine IgM. Selected chemical and physical characteris-

achieving full speed of 40,000 rpm; and immunoelectrophoretie patterns developed with anti-bovine IgM (upper trough) and anti-bovine gamma globulins (lower trough).

T E C H N I C A L NOTES

TaBra~ 1. Physical-chemical and chemical characteristics of bovine colostral Immunoglobulin. Property

Value

S~

18.2-

19.8

Y,~ 280nm

11.8 +

.2

Carbohydrate content Total hexoses Mannose Galactose Hexosamines Fucose Sialic acid

5.13 3.42 1.71 2.90 1.25 1.39

Sulfhydryl groups Total --SH gronps Free --SH groups S-S linkages

43.6 moles/mole .9 mole/mole 21/mole

+-_+ _ _ ± -.+

.12 .08 .08 .10 .05 .06

tics of three different IgM preparations are in Table 1. Extinction coefficients of IgM solutions in .15M NaC1 at concentrations of .0085 to .0857o followed Boyle's law with values ranging from 11.7 to 12.1 and averaging 11.8. These values are somewhat lower than 12.6 reported by Mukker and Froese (13) for their preparation of bovine IgM. Hexoses of IgM were primarily mannose and galactose in a ratio of 1.93:1. A value of 2:1 was used for calculation. In our preparations of IgM, hexose content ranged from 5.01 to 5.25 which is somewhat higher than that reported for human IgM (11) but lower than for bovine IgM (7). Calculated values for mannose and galactose suggested 3.42 ± .08~ mannose and 1.71 ± .04% galactose. Relatively high values for fucose were obtained in our study. In repeated analyses, fucose ranged from 1.2 to 1.37~ compared to .6 to .7 reported for human or bovine IgM (3, 11). Sialic acid ranging from 1.34 to 1.457o was similar to values for human IgM (11). Hexosamine content calculated as glueosamine ranged from 2.81 to 3.00% and averaged 2.90%. IgM contained only .9 moles of free -SH groups/mole of IgM (reel wt 900,000) indicating almost complete absence of free -SH groups. However, after reduction of S-S bonds, the total number of -SH groups was 43.6/mole of IgM. Thus, the number of S-S bonds would approximate 21/mole of IgM. To evaluate reliability of our method, -SH groups of fl-lactoglobulin were determined. This protein contained 1.8 moles of free -SH groups and 5.7 moles of total -SH groups per mole of protein based on a molecular weight

257

of 36,000 indicating 2 -SH and 2 S-S bonds/mole. Reported values for fl-lactoglobulin are 2 -SH groups and 2-3 S-S bonds. The presence of 21 moles of -SH/mole of IgM, although lower in value than that reported for human IgM, would be in accord with the five-subtmit theory for IgM discussed by Miller and Metzger (12), however. Similarity in structure between bovine serum and human IgM has been suggested by Beale and Buttress (1). Our results support the proposition that IgM from bovine colostrum is similar to its counterpart in bovine serum and human serum.

S. KUMAR and E. M. MIKOLAJCIK, Department of Food Science and Nutrition, Ohio Agricultural Research and Development Center, Columbus 43210

References

(1) Beale, D., and N. Buttress. 1972. Structural studies on bovine immunoglobulin. M. Biochem. Biophys. Aeta., 257:372. (2) Boas, N. F. 1953. Method for the determination of hexosamines in tissues. J. Biol. Chem., 204:553. (3) Cohen, S., and C. Milstein. 1967. Struc~ro and biological properties of immunoglobulins in Advances in Immunology. Edited by F. J. Dixon and H. G. Kunkel. Vol. 7, Academic Press, New York. (4) Davis, B. J. 1964, Disc electrophoresis. II. Method and application to human serum proteins. Int. Arch. Allergy, 38:618. (5) Dische, Z., and L. B. Shettles. 1948. A specific color reaction of methylpentose and a spectrophotometric mieromethod for their determination. J. Biol. Chem., 175: 595. (6) Ellman, G. L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys., 82:70. (7) Gough, Patricia M. 1966. Further studies of a rnereaptoethanol-sensitive Brucella agglutinin in bovine milk. Ph.D. dissertation, University of Minnesota, St. Paul. (8) Heimer, R., L. G. Clark, and P. H. Maurer. 1969. Immunoglobulins o~ sheep. Arch. Biochem., 131:9. (9) Kumar, S., and E. M. Mikolajcik. 1971. Isolation and partial characterization of bovine eolostral immunoglobnlins. J. Dairy Sci., 54: 753. (Abstr.) (10) Kumar, S., and E. M. Mikolajcik. 1972. Electrophoretic, irnmunoelectrophoretic, and ultracentrffugal characterization of proteins in whey fractions prepared by salt fractionation. J. Dairy Sci., 55:1237. (11) Miller, F., and H. Metzer. 1965a. Characterization of a human macrog|obulin. I. The molectdar weight o~ its subunit. J. Biol. Chem., 240:3325. (12) Miller, F., and H. Metzger. 1965b. CharacterJOURNAL OF DAIRY SOENCE VO~. 56, No, 2

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ization of a human macroglobulin. II. Distribution of the disulfide bonds. J. Biol. Chem., 240:4740. (13) Mukkur, T. K. S., and A. Froese. 1971. Isolation and characterization of IgM from bovine colostral whey. Immunochemistry, 8:257.

(14) Reisfeld, R. A., V. ]. Lewis, and D. E. Williams. 1962. Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature, 216:328; (15) Warren, L. 1959. The thiobarbituric acid assay of sialie acids. J. Biol. Chem., 208: 1971.

Blood Fatty Acids and Glycerol Response to Diet and Norepinephrine1 Abstract

Twelve mid-lactating Holstein cows divided equally into two groups were given either a normal diet or restrictedroughage high grain diet. During the 5th week, blood plasma glycerol and free fatty acids response of each group to 3.75 milligrams L-norepinephrine or saline were compared at 0, 10, 20, 30, and 40 minutes after intravenous injection. Mean control (saline) values (micromolar) for cows fed normal and restricted-roughage high grain diets were glycerol, 95 and 102 and free fatty acids, 394 and 467. Following norepinephrine injection, mean values (micromolar) for cows were glycerol, 112 and 127 and free fatty acids, 507 and 523. Glycerol response to norepinephrine was greater and free fatty acids apparently lower in cows fed restricted-roughage high grain diets than cows fed a normal diet. Diet appeared to alter capacity of adipose tissue to respond to norepinephrine. Feeding the restricted roughage-high grain depressed milk fat from 3.8 to 2.65 without affecting milk production.

motes accumulation of depot fat (3, 9), and increases esterifieation of fatty acids in adipose tissue (2). Lactating cows fed RR-HG revert toward a nonlactating state and hypothetically would resist mobilization of fatty acids in response to norepinephrine as in dry cows (12). Examination of this hypothesis is reported here. Materials and Methods

Twelve lactating Holstein cows in mid-lactation were equally divided into two groups on basis of milk production and stage of lactation. For 5 wk one group was fed a normal diet of corn silage, 11.4 kg; alfalfa haylage, ad libitum; and grain mixture, 1 kg/3 kg milk per head per day. The other group received RRHG diet of alfalfa haylage, 3.6 kg, and grain mixture, ad libitum. Percent composition of grain mixture was ground corn, 75.0; dehulled soybean meal, 15.0; molasses, 7.5; diealcium phosphate, 1.0; trace mineral salt, 1.0; and calcium carbonate, .5. Vitamins A and D were included in grain mixture at 2,200 international units (IU) per kg each. Average feed consumption (kg/day) of cows fed the normal diet was alfalfa haylage, 15.9; corn silage, 11.4; and grain mixture, 6.7. Cows fed RR-HG consumed on the average 3.6 kg alfalfa hayIntroduction lage and 12.9 kg grain mixture. Milk producInitiation of lactation frequently results in tion was recorded daily. Milk fat was deterloss of body weight, negative energ0r balance, mined weekly (Babcock). In the beginning of the last trial week, polyand mobilization of fat depots (4, 6). During lactation the concentration of blood plasma vinyl catheter was placed in a jugular vein of free fatty acids (FFA) and glycerol are in- each cow. Catheter patency was maintained creased by inhibiting esterillcation of fatty acids with 3.5% sodium citrate in saline (.85% and increasing active form of hormone-sensi- NaC1). On the next 2 consecutive days after tive or fat-mobilizing lipase in adipose tissue catheterization, saline (5.0 ml) and L-nore(12). On the other hand, feeding of restricted- pinephrine (3.75 mg in 5.0 ml saline) were roughage, high-grain (RR-HG) diet to lactat- injected intravenously over 5 min in each ing cows depresses milk-fat secretion (14), pro- cow in alternate sequence (12). Blood sampies were drawn at 0, 10, 20, 30, and 40 min after start of injection to determine plasma Received for publication August 15, 1972. 1Michigan Agricultural Experiment Station FFA and glycerol (12). Glycerol and FFA data were analyzed by Journal Article 6026, partially supported by Naanalysis of variance for repeat measurements tional Institutes of Health Grant AM 13177. JOURNAL OF DAtRY SCIENCE VOL. 56, NO. 2