SOIx4E CHARA E'ERISTICS OF THE FRACTCIGLQBULEE‘Q FRACTEQN OF [BC-VINE MILK “rests {or Hm Degree 0? N4. 5. MICEESAN STATE UNIVERSETY Otomars Veiss 1961 LIBRARY Michigan State University SOME CHARACTERISTICS OF THE LACTOGLOBULIN FRACTION OF BOVINE MILK By Otomars Veiss A THESIS Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1961 “MIX, \J N ii ACKNOWLEDGMENTS The author wishes to express his appreciation to Dr. 'J. Robert Brunner, Professor of Food Science, for directing this re- search and for his suggestions and counsel. The author also acknowledges the technical advice and efforts of Dr. M. P. Thompson in several phases of this research. Grateful acknowledgment is due to the Michigan State Univer- sity and the Michigan Agricultural Experiment Station for the funds and facilities which made this research possible. TABLE OF CONTENTS Procedures for the Isolation of Lactoglobulins The fractionation of bovine milk with ammonium sulfate The fractionation of bovine colostrum with ammonium sulfate The fractionation of bovine milk and colostrum with sodium sulfate The fractionation of bovine milk and colostrum with Rivanol Analytical Methods Electrophoresis Ultracentrifugal analysis Nitrogen determination Carbohydrate determination Fractionation of Milk Proteins on DEAE-Cellulose Columns Preparation of the adsorbent column Preparation of the sample Development of the chromatOgram Examination of the effluent EXPERIMENTAL RESULTS ................................. Isolation of Lactoglobulins Fractionation of Lactoglobulins on DEAE-Cellulose Columns Analysis of Fractions Nitrogen determination Ultracentrifugal characteristics Carbohydrate content Electrophoresis 11 12 12 12 12 l3 13 14 19 19 19 20 20 20 20 20 iv Page DISCUSSION .................................................. 31 Isolation of Lactoglobulins 31 Fractionation of Lactoglobulins on DEAE-Cellulose Columns 32 Analysis of Fractions 34 Nitrogen content 34 Electrophoretic mobilities 34 Ultracentrifugal characteristics 34 Carbohydrate content 35 SUMMARY .................................................. 36 CONCLUSIONS ............................................... 38 REFERENCES CITED ........................................ 4O Table II. III. IV. LIST OF TABLES Nitrogen content of lactoglobulin fractions of bovine milk and colostrum ........................................ Electrophoretic mobilities of lactoglobulin fractions of bovine colostrum in veronal buffer, pH 8. 6, F/2 = O. 1, athC .. Sedimentation-velocity coefficients (820) for lactoglobulins of bovine colostrum in veronal buffer, pH 8. 6, F/Z = O. 1 . . Carbohydrate content of the lactoglobulins of bovine colostrum Page 27 28 29 30 Figure 10. LIST OF FIGURES A schematic diagram showing the fractionation of bovine milk with ammonium sulfate ........................... A schematic diagram showing the fractionation of bovine colostrum with ammonium sulfate ...................... A schematic diagram showing the fractionation of bovine milk and colostrum with sodium sulfate .................. A schematic diagram showing the fractionation of bovine milk and colostrum with Rivanol ........................ Gradient elution diagram of whey proteins from a DEAE- V cellulose column ............... , ...................... Gradient elution diagram of lactoglobulins from a DEAE- cellulose column ...................................... Gradient elution diagram of pseudoglobulin from a DEAE- cellulose column ...................................... Gradient elution diagram of euglobulin from a DEAE- cellulose column ...................................... Electrophoretic patterns of lactoglobulin fractions in veronal buffer, pH 8. 6, F/Z = 0.1 ..................... The ultracentrifugal diagrams of lactoglobulin components . vi Page l6 17 18 21 22 23 24 25 26 INTRODUCTION The classical lactoglobulin fraction of colostrum and milk has been obtained by saturating whey with magnesium sulfate or by half- saturation with ammonium sulfate. In general, lactoglobulin refers to the slowest electrophoretic components in colostrum and milk. The American Dairy Science Association Committee on the nomenclature, classification, and methodology of milk proteins (Jenness e_t_a_l_. , 1956) tabulated a mobility ranging from - l. 8 to -2. 2 x 10-5 (cm. v...1 sea-l) to this fraction. Upon prolonged dialysis the lactoglobulin separates into a water soluble pseudoglobulin and a water insoluble euglobulin; pseudo- globulin remains in solution and the euglobulin precipitates out. These proteins have often been called the ”immune lactoglobulins" because they were associated with antibody activity (Smith, 1946a, b). However, Pierce (1955) has shown that these components were present in milk in the absence of specific immunization. In addition to their immune acti- vity they have been reported to contain carbohydrates. Advancements in techniques used in the fractionation and purification of proteins have made it possible to demonstrate the presence of new components in protein fractions which once were considered as homo— geneous. The general assumption has prevailed, based on ultracentrifugal evidence, that the pseudoglobulins and euglobulins are heterogeneous protein fractions. The objective of this study was to isolate electrophoretically "homogeneous" lactoglobulin components and to characterize the constituent conjugated carbohydrate moiety. REVIEW OF LITERATURE Eugling (1880) was the first to recognize globulin in milk. He reportedthat following prolonged treatment of diluted colostrum whey with carbonic acid white flakes separated out which were soluble in five per cent salt solution. Therefore he considered these flakes to be globulin. A similar suggestion was made by Hammersten (1883) who observed that after separating casein from milk the filtrate contained albumin and a substance separated by saturation with magnesium sulfate. His experi- ment led him to suggest that the precipitate was a globulin. The name ”lactoglobulin" was introduced by Sebelien (1885) who obtained a flocculent precipitate by saturating whey with magnesium sul- fate. This protein fraction appeared to be identical in characteristics with serum globulin. Further, he found an abundance of lactoglobulin in bovine colostrum. Halliburton (1890) denied the existance of globulin in milk and regarded the isolated substance as lactalbumin. However Sebelien (1891) proved that albumin was not precipitated by saturating whey with magnesium sulfate, but remained in solution from where it could be precipitated by the addition of acetic acid. Storch (1897) used sodium sulfate at room temperature to isolate the globulins from milk by saturating it with the salt, which, at room tem- perature, was equivalent to a 14-19 per cent solution. Schlossmann (1896- 1897) separated globulin by saturating milk with magnesium sulfate and observed that the globulin, after prolonged standing, collected on the surface of the liquid. Simon (1901) re-examined the 3 procedures of Sebelien (1891) and Schlossmann (1896-1897) and obtained globulin fractions which proved to be similar in their composition and solubility. Bauer and Engel (1911) compared colostrum and milk and found that globulin was more active than albumin in building antibodies. They ob- served no biological difference between milk and colostrum. The same was found to be valid for the proteins of blood serum compared to those of milk and/or colostrum. Crowther and Raistrick (1916) reported that milk globulin, like blood globulin, could be separated into euglobulin and pseudoglobulin. They precipitated globulin with magnesium sulfate, re- dissolved it in distilled water, and dialyzed it for six days in running water. The globulin was thus separated into two fractions; euglobulin-- insoluble in water. and pseudoglobulin--soluble in water. Dudley and Woodmann (1918) and Woodmann (1921) investigated euglobulin and pseudoglobulin and found that these proteins were struc— turally identical. Racemization was employed to establish identity or non-identity of related proteins. They investigated the optical rotational properties of euglobulin and pseudoglobulin in alkaline solution and also made a comparative study of the optical properties of amino acids derived from the hydrolysis of racemized euglobulins and pseudoglobulins. Howe (1922) precipitated globulins from colostrum with sodium sulfate and considered the material precipitated up to 14. 2 per cent at 340 C. to be euglobulin; that at 14. 2 - l8. 4 per cent as pseudoglobulin I and casein (casein was obtained from the filtrate by acidification); that at 18. 4 - 21. 5 per cent as pseudoglobulin II. This last fraction was re- covered in small yields and there was no positive evidence that this was a separate protein. Howe (19 21) obtained these fractions by following an isolation scheme he employed to fractionate blood proteins. Smith (1946a, b; 1948) made a series of outstanding studies and contributions to our knowledge of the lactoglobulin fraction. He found that the globulin precipitated by magnesium or ammonium sulfate was a mixture of proteins. He devised a scheme for isolating an electrophor- etically homogeneous globulin fraction by fractionation with ammonium sulfate. However studies in the ultracentrifuge revealed that all of the isolated lactoglobulins contain more than one fraction. The globulin character of the isolated protein fractions was indicated by their preci- pitation at low concentrations of ammonium sulfate, low solubility near the isoelectric point, and by marked increase in solubility in the presence of neutral salts. Smith reported that exhaustive dialysis of the lacto- globulin fraction resulted in the separation of euglobulin (water insoluble) and pseudoglobulin (water soluble) fractions and that immune activity was associated with both of these proteins. Further, he showed by electro- phoretic analysis that the lactoglobulin of milk and colostrum were iden- tical. An elemental analysis of lactoglobulins by the same investigator revealed an absence of phosphorus, but showed the presence of sulfur and carbohydrates. Smith and Greene (1947) reported that lactoglobulins had a high threonine content, and that cystine and methionine accounted for the sulfur of these proteins. The total carbohydrate content of lactoglobulins in milk and colostrum was contributed from the protein bound hexose and hexosamine (Smith, Greene and Bartner, 1946; Smith, 1946b). Smith (1946b) reported the values of pH 5. 6iand 6. 05 for the isoelectric points of pseudoglobulin and euglobulin, respectively. Smith (1946a, b) was the first to use electrophoresis and ultracen- trifugation as well as chemical analysis in comparing the properties of lactoglobulin fractions. He records electrophoretic mobilities for pseudo- globulin and euglobulin of milk and colostrum of —2. 5 and -1. 7, and -2. 2 and -1. 9, respectively. Murthy and Whitney (1958) reported values of -2. O4 and -l.76 for similar protein fractions. The sedimentation coefficient of the principal component (about 84 - 89%) of colostrum and milk was reported to be approximately 520’ w=7 (Smith, 1946a, b). The second most concentrated component had a value of 10 Svedberg units. A third component, with a sedimentation coefficient of about 20 Svedberg units, was found only in the euglobulin fraction. He also reported a component of 2 to 3 Svedberg units in the pseudoglobulin fraction of colostrum. Deutch (1947): reported somewhat lower values and Murthy et_a_l_. (1958) higher values for similar protein fractions. A new method for isolating lactoglobulins from milk and colostrum was introduced by Kenyon, Anderson and Jenness (1959), as an adaptation of a method developed by Horejsi and Smetana (1956) for the isolation of gamma-globulin from blood serum. In this method Rivanol (2-ethoxy-6, 9- diamino-acridine lactate) was used to form a metal-combining globulin complex which remained in solution. The precipitated proteins were in}? filtered off. Rivanol was removed from the supernatant by adsorption on activated charcoal, leaving a crude lactoglobulin fraction in solution. EXPERIMENTAL PROCEDURE Procedures for the Isolation of Lactoglobulins Electrophoretically homogeneous euglobulin and pseudoglobulin fractions were separated from the lactoglobulin of milk and/or colostrum by previously reported methods and include: a. fractionation of bovine milk and colostrum with ammonium sulfate (Smith, 1946a, b); b. fractionation with sodium sulfate (Howe, 1922); c. fractionation by the use of Rivanol (Kenyon et al. , 1958). The fractionation of bovine milk with ammonium sulfate. The scheme for the isolation and fractionation of the lactoglobulin is shown in Figure 1. Fresh raw milk was separated three times at 400 C. in a laboratory-size cream separator. The skimmilk was acidified with 0. 1 N HCl to pH 4. 6 and the casein removed by filtration. The acid whey was adjusted to pH 6. 5 with 0. 1 N NaOH and ammonium sulfate added to 0. 5 saturation to precipitate the crude globulin (Fraction A). Fraction A was redissolved to about three per cent protein concentration, the pH adjusted to 4. 6 and ammonium sulfate added to 0. 25 saturation. The ensuing precipitate (Fraction 9) was removed by centrifuging in a Servall centrifuge for 15 minutes at 25, 000 xG. The supernatant was filtered through a thick layer of glass wool. The lactoglobulins (Fraction _D_) were precipitated from the supernatant at 0. 4 saturation with ammonium sulfate at pH 6. 0. This fraction was reworked by dissolving in distilled water to about three per cent protein concentration, adjusting pH to 4. 5 and filtered. Ammonium sulfate was added to O. 3 saturation and Fraction _E_ was precipitated. The supernatant was brought to pH 6. O and ammonium sulfate added to O. 4 saturation to obtain the precipitate (Fraction 1:). Precipitates were re- dissolved and dialyzed free of salt against distilled water. The lactoglo- bulin fractions were then dried by lyophilization. The fractionation of bovine colostrum with ammonium sulfate. The scheme for fractionation and isolation of lactoglobulins from colostrumis shown in Figure 2. After separating the fat from the colostrum the skim- milk was diluted fivefold with distilled water and slowly adjusted to pH 4.5 with 0. 5 N HCl. The casein precipitate (Fraction _A_) was removed by filtration. The lactoglobulins were precipitated at pH 6. 0 by adjusting the whey to 0. 3 saturation with ammonium sulfate (Fraction _B_). Ammonium sulfate was added to the supernatant to O. 5 saturation and Fraction _C_ was precipitated. Fractions _B_ and 9 were redissolved in distilled water to about five per cent protein concentration, cleared of residual undissolved material by filtration, and reprecipitated at the same limits of salt con- centration. The lactoglobulins were redissolved and dialyzed against distilled water. The pseudoglobulin and euglobulin fractions were lyophilized. The fractionation of bovine milk and colostrum with sodium sulfate. The scheme for the isolation and fractionation of lactoglobulins is shown 0 in Figure 3. Fresh raw milk was separated three times at 40 C. The skimmilk was acidified with O. 1 N HCl to pH 4. 6 and the casein removed by filtration. The acid whey was adjusted to pH 6. 5 and 14. 2 per cent with respect to sodium sulfate, added at 340 C. to precipitate the euglo- bulin fraction. The precipitated fraction was collected by centrifugation, redissolved and dialyzed free of salt prior to lyophilization. Sodium sulfate was added to the supernatant to increase the salt concentration to 18. 4 per cent. The precipitated pseudoglobulin was collected by cen- trifugation, redissolved, dialyzed salt free and lyophilized. The fractionation of bovine milk and colostrum with Rivanol. The scheme of isolation and fractionation of lactoglobulins is outlined in Figure 4. Fresh raw milk was separated three times at 400 C. The skimmilk was acidified with O. 1 N HCl to pH 4. 6 and the casein removedby filtration. To one volume of the acid whey to be fractionated, 3. 5 volumes of 0. 4 per cent aqueous Rivanol solution was added. The solution was adjusted to pH 8. 5 to precipitate the lactoglobulins which were filtered off. ‘ Activated charcoal was added to remove the Rivanol by adsorption and filtered off. The clear filtrate, containing lactoglobulins, was pervapo— rated, dialyzed against distilled water. The euglobulins and pseudoglo- bulins were recovered and dried by lyophilization. Analytical Methods Electrophoresis. All electrophoretic data were obtained with a Perkin-Elmer Model 38-A Electrophoresis apparatus. Protein solutions were made up in veronal buffer, pH 8. 6, u = O. l and dialyzed for six hours using a magnetic stirrer or for seventy-two hours without a stirrer in 10 two veronal buffer changes of 500 milliliters at 20 C. The completion of dialysis was determined by measuring the specific conductivity of the protein solution. The specific conductivity was calculated from the following equation: cc K = "if where K - specific conductivity cc - cell constant R - resistance of solution in ohms observed at l0 C. Protein concentrations were determined after dialysis by determining the difference between the dry weight of the buffer and the dialyzed pro- tein solution. The electrophoretic mobilities were calculated using the following equation: -1 -l daK C . . 6C. : “(m V S ) itRm where d - distance migrated (cm.) a - cross sectional area of the cell (cm. ) K - specific conductivity cell constant (ohms) = O. 8491 i - current (amps) t - time (sec.) R - resistance of buffer (ohms) m - magnification factor = 1.1 The field strength was calculated from the following equation: 11 where F - field strength or potential gradient i - current (amps) . 2 a - cross sectional area of the cell (cm. ) K - specific conductivity of the buffer-protein solution Ultracentrifugal analysis. Sedimentation coefficients were deter- mined by the sedimentation-velocity technique, employing a Spinco Model E Centrifuge equipped with analytical accessories. The determinations were made at 200 C. , and veronal buffer at pH 8. 6 and ionic strength of O. l was used as a carrier for the protein solutions. Sedimentation coef- ficients were calculated from the following equation: Ad/m S : 2 r 2 4n (x +— )rps t m where S - sedimentation coefficient (sec.) Ad - distance migrated in unit time (cm.) m - magnification factor = 2.1 x - distance of refractive gradient from axis of rotation (cm. ) = 5. 72 2 411 - 39. 5 r - distance half-way between the peaks used for calculation(cm.) rps - revolutions per second t - time corresponding to observed position of sedimenting boundary indicated by subscripts (sec. ) 12 Nitrogen determination. The official A. O. A. C. (1950) micro- Kjeldahl method was used to measure the nitrogen content of the various protein fractions. Carbohjdrate determination. Protein bound carbohydrates were determined according to procedures outlined by Glick (1955). Hexose was determined by means of an orcinol method for which galactose served as a standard. Hexosamine was determined on the basis of the reaction observed with galactosamine when used as a standard. Authentic fucose was employed as a standard for the fucose determination. Diphenylamine was employed in the determination of protein bound neuraminic acid. Orosomucoid, containing 11. 2 per cent neuraminic acid, was employed as a secondary standard for neuraminic acid. Fractionation of Proteins on Diethylaminoethyl-Cellulose (DEAE) Columns In general the fractionation procedure developed by Rackis, Sasame, Anderson, and Smith (1959) for chromatography of soybean proteins on a DEAE-cellulosic column was followed. Preparation of the adsorbent column. The cellulosic anion-exchange adsorbent (DEAE) was suspended in water, stirred and titrated to pH 7. 6 with a concentrated solution of dihydrogen phosphate. The adsorbent was washed several times on a Buchner funnel with 0. 01 M sodium phosphate buffer at pH 7. 6 and re-suspended in the same buffer. The fines were decanted from the suspension after settling for 30 minutes. Following equilibration for at least 12 hours with the buffer, a slurry of the adsorbent 13 was poured into the exchange column, equipped with a fritted disc at the bottom, and allowed to settle until a constant column height was achieved. The packed column was connected to a fraction collector and washed with several volumes of the phosphate buffer. The column assembly used was similar to that described by Hirsh and Ahrens (1958). The packed column had dimensions of 16 - 17 x l. 5 cm. Following the completion of a chromatographic separation, the adsorbent was removed from the column and washed with l N NaOH until free from protein. This was checked by addition of 10 per cent TCA solution to equal volume of wash water following the regeneration of the adsorbent. The regenerated adsorbent was packed as described above. Preparation of the sample. Samples containing 300 milligrams of lyophilized protein in 10 milliliters of 0. 01 M sodium phosphate buffer at pH 7. 6 were poured onto the column and allowed to enter the adsorbent under flow conditions induced by gravity. The walls of the column were washed down with 5 milliliters of the buffer, followed by pouring 5 milli- liters of buffer on top of the column before elution was started. Development of the chromatogram. Gradient elution was carried out at room temperature and NaCl (0 --- O. 345 M) in 0. 01 M sodium phosphate buffer at pH 7. 6 was used to develop the chromatOgrams. Ten milliliter fractions were collected at a rate of 60 to 70 milliliters per hour with an automatic collector equipped with a 10 milliliter syphon. The fractions which compriseddiscrete peaks were combined, dialyzed, lyophilized and subsequently analyzed. 14 Examination of the effluent. The absorbance of each effluent frac- tion was determined in a Beckman DK-2 Spectrophotometer at 278 mg. The fractions comprising discrete peaks were combined, dialyzed free of salt against distilled water, lyophilized and subsequently analyzed. WHOLE RAW MILK 0 [Separate 3x at 40 C. SKIMMILK Acidify with 0.1 N HCl to pH 4. 6 Filter [— 1 CASEIN WHEY (Discard) Adjust pH to 6.5 with 0.1 N NaOH Add (NH4)2804 to 0.5 saturation. Centrifuge at 2000 xG, 15 min. PRECIPITATE (FRACTION A) SUPERNATANT Eude globulin (Discard) Redissolve at 3% protein conc. Adjust pH to 4. 6. Add (NH412 504 to 0.25 saturation. Centrifuge at 25000 xG, 15 minutes. PRECIPITArTE (FRACTION _c_:_) SUPERNIATANT (Discard) Filter through glass wool. Adjust to pH 6. 0. Add (NH4)2 $04 to 0. 4 saturation. Centrifuge at 25000 xG, 15 min. PRECIPITATERFRACTION _D) SUPERNATANT Redissolve at 3% protein conc. (Discard) Adjust to pH 4. 5. Filter off insoluble residue. Add (NH4)ZSO4 to O. 3 saturation. Centrifuge at 25000 xG, 15 min. PRECIPITATE '(FRACTION E) SUPERNATANT IRedissolve in water. Adjust to pH 6. 0. IDialffze. Add (NH4)Z SO4 to 0. 4 saturation. PRECI'PITATE SUPERNTATANT (Discard) (Pseudoglobulin) I l PRECIPITATE (FRACTION _F_) SUPERNATANT Redis solve in water. (Discard) Dialyze. PRECIPITATE SUPERNATANT (Euglobulin) (Pseudoglobulin) Figure 1. A schematic diagram showing the fractionation of bovine milk with ammonium sulfate. l6 COLOSTRUM lSeparate 3x at 400C. r . FAT SKIMMILK (Discard) Dilute fourfold with distilled water. Adjust to pH 4. 5 with O. 5 N HCl. Filter. I i j CASEIN (FRACTION A) WHEY (Discard) Adjust to pH 6. O with 0.5N NaOH. Add (NH4)ZSO4 to 0.3 saturation. Centrifuge at 2000 xG, 15 min. W i I PRECIPITATE (FRACTION _B_) SUPERNATANT Redissolve in water to 5% conc. Add (NH4)ZSO4 to 0. 5 Add (NH4)ZSO4 to 0.3 saturation. saturation. Centrifuge at 20000 xG, I . PRECIPITATE SUPERNATANT 15 mmuteS- (Euglobulin) (Pseudoglobulin) PRECIPITATE|(FR.ACTION C) SUPER- Redissolve in water to NATANT 5% concentration. (Discard) saturation. Centrifuge at 20000 xG 15 minutes. PRECIPITATE SUPERNATANT (Lactoglobulins) (Discard) Redis solve in water. Dialyze. l j PRECIPITATE SUPERNATANT (Euglobulin) (Pseudoglobulin) Figure 2. A schematic diagram showing the fractionation of bovine colostrum with ammonium sulfate. WHOLE RAW MILK Separate 3x at 400 C. l7 EXT SKIMMILK (Discard) Acidify to pH 4. 6 with 0.1 N HCl. Filter. ,CASrEIN WHEY (Discard) Adjust to pH 6.5 with 0.1 N NaOH. Add 14.2% NaZSO4 at 34° C. Centrifuge at 20000 xG, 15 min. FL PRECIPITATE (Euglobulin) Redis solve in water. SUPERNATANT _. Add NaZSO4 to 18.4% at 340C. Centrifuge at 20000 xG, 15min. * I Dialyze. . I ' Lyophilize. PRECIPITATE SUPERNATANT (Pseudoglobulin) (Discard) Redissolve in water. Dialyze. Lyophilize. Figure 3. A schematic diagram showing the fractionation of bovine milk and colostrum with sodium sulfate. 18 WHOLE RAW MILK Separate 3x at 400 C. r FAT SKIMMILK (Discard) Acidify to pH 4. 6 with 0. l N HCl. Filter. IL CASEIN WHEY (Discard) Add 3. 5 volumes of 0.4% aqueous Rivanol solution to 1 volume of whey. Adjust to pH 8. 5. Filter. h PRECIPITATE SUPERNATANT (Discard) Add activated charcoal. Filter. PRECIPITATE SUPERNATANT (Discard) (Lactoglobulins) Pervaporate. Dialyze. r I PRECIPITATE SUPERNATANT (Euglobulin) (Pseudoglobulin) Lyophilize. Lyophilize. Figure 4. A schematic diagram showing the fractionation of bovine milk and colostrum with Rivanol. l9 EXPERIMENTAL RESULTS Isolation of Lactoglobulins The best isolations of lactoglobulins based on electrophoretic homo- geneity were obtained by following the procedure of Smith (Figure l) in which ammonium sulfate was used as the salting-out agent. To obtain workable amounts of electrophoretically homogeneous lactoglobulins, this fractionation procedure as shown in Figure l was adapted and used for the fractionation of colostrum. The free-boundary electrophoretic patterns of the fractions isolated are shown in Figure 9; their electrophoretic mobilities are listed in Table II. Fractionation of Lactoglobulins on DEAE—Cellulose Columns The elution diagram of acid whey proteins from the DEAE cellulosic column is shown in Figure 5. Figures 6, 7 and 8 show the elution diagrams of electrophoretically homogeneous lactoglobulin, pseudoglobulin and eu- globulin fractions respectively (Figure 9). The reproductability of the position and height of the chromatographic peaks were good. Identical elution patterns were obtained in all cases. In Figure 6, area under peak 1 consists of proteins collected in tubes 3 to 9; area under peak 2 of effluent collected in tubes 12 to 22; area under peak 3 of effluent in tubes 23 to 50. In Figure 7, area under peak 1 con- sists of effluent collected in tubes 4 to 12, and area under peak 2 of effluent in tubes 14 to 53. In Figure 8, area under peak 1 consists of effluent in tubes 3 to 14; area under peak 2 of effluent in tubes 15 to 57. F “.mn _ 20 Analysis of Fractions Nitgogen determination. The results of the Kjeldahl nitrogen deter- minations for lactoglobulin fractions of milk and colostrum are listed in Table I. The concentration of proteins was calculated on a dry weight basis. Ultracentrifugal characteristics. Sedimentation-velocity studies were made on lactoglobulin, pseudoglobulin, euglobulin, and pseudo- globulin Fraction 2 from the DEAE-cellulose column. The sedimentation- velocity diagrams for these fractions are shown in Figure 10. Table III lists the sedimentation-velocity coefficients for all components in the above listed fractions. Carbohydrate content. Protein bound hexose, hexosamine, fucose and neuraminic acid concentrations of the lactoglobulin fractions and their components are recorded in Table IV. The results were calculated on a dry weight basis. Electrophoresis. 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Br taillight?” .CESHOU omoddflooumaoqmfl .m Scum cmadfionSo mo Eduwdwp €033? “£66320 .w ondmwm 4 2 :8; mzbqo> Hzmoqmmm ETOS ”N 833$ 37% g .83qu com com cos 000 com 2:. com com 2: K . I I). a a. o -N . .m a. A. -m s i -w -m 3 Lo . m r o" o' 0' c5 c5 iIu1 8L2 .LV ADNVEIHOSHV Ascending Descendi_ng_ Lactoglobulin Fraction: 8800 sec. 12.1 volt cm.-1 Pseudoglobulin Fraction: 8100 sec. 12.1 volt cm.’1 Euglobulin Fraction: 8700 sec. 12.1 volt cm.-1 Figure 9. Electrophoretic patterns of lactoglobulin fractions in veronal buffer, pH, r/z = o. 1. (A) 59, 780 rpm Conc. , l. 5% Bar angle, 650 Temp. , 20°C. Veronal, pH 8.6, p = 0. 1 Time in minutes (C) 59, 780 rpm Conc. , 1. 2% Bar angle, 600 Temp., 20° c. Veronal, pH 8. 6, p. = 0. l 68 Time in minute 3 (E) 59, 780 rpm Conc. , 1. 0% Bar angle, 600 Temp., 20° c. Veronal, pH 8.6 p. = 0. l Time in minute 5 26 (B) 59, 780 rpm Conc. , l. 5% Bar angle, 650 Temp., 20° c. Veronal, pH 8.6, p = 0.1 50 Time in minutes (D) 59, 780 rpm Conc. , 1. 0% Bar angle, 600 1 Temp., 20° c. A; Veronal, pH 8.6, - p. = 0. l 54 Time in minutes (F) 59, 780 rpm Cone. , 1. 0% Bar angle, 600 Temp., 20° c. Veronal, pH 8.6, p. = 0. l Time in minute 5 Figure 10. The ultracentrifugal diagrams of: (A) lactoglobulin, (B) pseudo- globulin, (C) pseudoglobulin fraction 2 from DEAE-cellulose column, (D) supernatant of dialyzed pseudoglobulin fraction 2 from DEAE-cellulose column, (E) precipitate of dialyzed pseudoglobulin fraction 2 from DEAE-cellulose column, (F) pseudoglobulin from DEAE-cellulose column minus fraction 2. x denotes protein ofa slow sedimenting component which could not be measured. 27 TABLE I Nitrogen content of lactoglobulin fractions of bovine milk and colostrum Protein fractiona Nitrogen content (‘70) Milk Lactoglobulin 15. 72 Pseudoglobulin 15. 58 Euglobulin 15. 83 Colostrum Lactoglobulin 15. 68 Pseudoglobulin 15. 16 Euglobulin 16. 07 a . . . . Obtained by salting out With ammonium sulfate. 28 TABLE II Electrophoretic mobilities of lactoglobulin fractions of bovine colostrum in veronal buffer, pH 8.6, r/z = 0.1, at 1° c. b Ele ctr ophor etic mobility (p) Protein Concentration fractiona (‘70) Descending Ascending Lactoglobulin l. 5 -1. 96 - 2. 13 Pseudoglobulin l. 5 -1. 93 - 2. 09 Euglobulin 0. 8 -1. 68 -1. 75 a . . . . Obtained by salting out With ammonium sulfate. - -1 _ bu = cm.2 v. 1 sec. x10 5 29 TABLE III Sedimentation-velocity coefficients (820) for lactoglobulins of bovine colostrum in veronal buffer, pH 8. 6 F/Z = 0.. Sedimentation-velocity coefficients (S " s 10-13 ) Protein fraction Concentration 20 - 20 X sec. (070) ----------- Peaks ------------ 1 2 3 Lactoglobulina' b l. 5 6. 10 9. 50 20. 63 , a, b Pseudoglobulin l. 5 6. 83 9. 68 - Pseudoglobulin fraction 2 from DEAE-cellu- lose columnb l. 2 6. 15 9. 34 - Supernatant of the dialyzed pseudo- globulin fraction 2 from DEAE- cellulose column 1. 0 6. 43 - — Precipitate of the dialyzed pseudo- globulin fraction 2 from DEAE- cellulose column 1. 0 6. 72 10. O6 - Pseudoglobulin from DEAE-cellulose column without fraction 2b 1. o 6. 81 - - Euglobulina' b 0.7 7. 11 9. 85 20. 86 a . . . Obtained by salting out With ammonium sulfate. These fractions contain one very slow sedimenting component which could not be measured. TABLE IV Carbohydrate content of the lactoglobulins of bovine colostrum Carbohydrate (mg. /100 mg. protein) Protein fraction Hexos Neur- Hexose . Fucose aminic Total amine _ ac1d Lactoglobulina 0.71 1. 50 0.54 1. 66 4. 41 Lactoglobulin from DEAE- cellulose column (Figure 6) Fraction 1 1.42 1.93 0.61 1.81 5.77 Fraction 2 1. 96 Z. 28 O. 75 Z. 27 7. 26 Fraction 3 l. 50 2. 43 0. 84 2. 57 7. 34 Pseudoglobulina 0.84 2. 15 0.73 2. 12 5. 84 Pseudoglobulin from DEAE- cellulose column (Figure 7) Figure 1 1. 20 3.15 0. 98 3.18 8. 51 Figure 2 1.44 1.15 0.49 1.51 4.59 Euglobulina 3. 24 2. 56 o. 86 2. 8o 9. 46 Euglobulin from DEAE- cellulose column (Figure 8) Fraction 1 3. 90 3. 00 0. 89 3. 03 10. 82 Fraction 2 2. 90 2. 40 0. 77 2. 34 8. 41 a . . . Obtained by salting out With ammonium sulfate. 31 DISCUSSION In this study the term "lactoglobulins" was applied to the components of colostrum with an electrophoretic mobility ranging from - 1. 8 to -2. 2. Is olation of Lactoglobulins In the comparison of the four different methods of fractionation and '3! isolation of lactoglobulins, the best results were obtained by the procedure of Smith (1946b), as outlined in Figure 1. Fraction 1: which represents the lactoglobulin appeared to be electrophoretically homogeneous. Pseudo- '. J it... globulin and euglobulin fractions obtained from Fraction _F_ upon dialysis showed electrophoretically homogeneous characteristics. The pseudo- globulin, Fraction _E_, was also electrophoretically homogeneous. How- ever, the yields of these proteins from bovine milk were too small, and large quantities of milk had to be used to obtain sufficient amounts for further studies. In the isolation of lactoglobulins from colostrum by salting-out with ammonium sulfate (Figure 2), only the pseudoglobulin fraction showed electrophoretically homogeneous characteristics. The lactoglobulin and the euglobulin fractions showed several peaks in their electrophoretic patterns. The euglobulin fraction was pinkish—gray in its appearance suggesting the presence of other proteins. The sodium sulfate fractionation procedure as outlined in Figure 3 did not produce homogeneous preparations. The Rivanol fractionation procedure (Figure 4) was simple to perform 32 and the fractions obtained were electrophoretically homogeneous. However, it was not possible to remove completely the clarifying charcoal which was carried in the lyophilized fractions and therefore obviating further analytical analysis. This method offers a simple procedure for obtaining lactoglobulins if a substance other than charcoal could be employed for the removal of the Rivanol. To obtain sufficient quantities of electrophoretically homogeneous components, the lactoglobulins were prepared from colostrum by the method outlined in Figure 1. The lactoglobulin preparation designated Fraction _1': was used throughout this study. Figure 9 presents the elec- trophoretic patterns of this fraction and its components. Fractionation of Lactoglobulin on DEAE-Cellulose Columns Studies in the analytical ultracentrifuge have revealed that the lacto- globulin preparations contained more than one component. In this phase of the study an attempt was made to separate as many components of the lactoglobulin fraction as possible. The chromatograms presented here (Figures 6-8) demonstrate the separation of lactoglobulins into constituent components by following their elution from a DEAE-cellulose column. The chromatogram for acid whey proteins (Figure 5) is in close agreement with the elution diagram presented by Yaguchi, Jennings, and Tarassuk (1959). Figure 6 shows the resolution of electrophoretically homogeneous lactoglobulin fraction into four distinct peaks, designated A to D in the order of their elution. 33 Similarly, Figure 7 shows the resolution of an electrophoretically homo- geneous pseudoglobulin fraction into five distinct peaks, designated A to E. Data. from this diagram suggest that areas under peaks _C_ and 2 were the main components of the pseudoglobulin fraction. Counterparts of these same peaks appear in the chromatogram for lactoglobulin (Figure 6). However, in this instance they appear as separate peaks, an observation attributed to the. smaller protein concentration. The small peak E in Figure 7 was reproducible in every fractionation and therefore evidenced to be a distinct component. In Figure 8 the electrophoretically homo- geneous euglobulin fraction was resolved into two distinct peaks (A and _B) and a relatively flat region (_C_). This region seems to cover many pro- teins or various states of aggregation of constituent proteins having similar affinities for this adsorbent. This flat region is masked by the two distinct peaks 9 and 2 of pseudoglobulin in the lactoglobulin chroma- togram (Figure 6). Area under peak _A_ in all chromatograms appears to be proteins not adsorbed on the column. A very sharp peak _B_ is present oniall chromatograms. An interesting observation was made upon prolonged dialysis of pseudoglobulin Fraction 2 from the DEAE-cellulose column. The fraction separated into a water insoluble (white precipitate) and a water soluble component. This suggested that one of the components plays the role of a stabilizer in the pseudOglobulin complex. It is also possible that the column partially denatured the pseudoglobulin complex. A similar observation was made with the lactoglobulin Fraction _2 from DEAE- cellulo s e column. 34 Analysis of Fractions Nitrogen content. The results in Table I are in close agreement with previously reported nitrogen values (Smith, 1946a, b). Electrophoretic mobilities. The mobility of lactoglobulin was approximately the same as observed by Smith (1946a), -2. 1; whereas that of pseudoglobulin was different from his value of -2. 2 (1946b). Murthy 31:31. (1958) report a value of -2. 04. The mobility of euglobulin conforms with the data reported by Murthy _e_t__a_l. (1958), - l. 76; and that reported by Smith (1946b), -1. 80. Ultracentrifugal characteristics. The data listed in Table 111 show that all of the protein preparations contained several ultracentrifugal sedimentation boundaries. The values observed for lactoglobulin were approximately the same as reported by Deutch (1947), namely, 6, 10 and 20 Svedberg units. Pseudoglobulin yielded two values which were in close agreement with Smith's (1946a) values of 7 and 10 Svedberg units. Murthy 131. (1958) reported 7. 69 and 10. 7 Svedberg units. The differ- ences can be accounted for by the different protein concentrations used in this study as well as the manner in which the experimental data were corrected. Euglobulin yielded three boundaries with sedimentation- velocity coefficients in close agreement with Smith's (1946b) values of 7, 10 and 20 Svedberg units but lower than those observed by Murthy (it 2;]: (1958), namely, 7. 93, 11. 85 and 22. 98 Svedberg units. The lacto- globulin fractions contained one slow sedimenting boundary which could not be measured, because it did not move far enough to make a 35 measurement. Smith (1946b) reported this particular component only in the pseudoglobulin fraction. The purified pseudoglobulin, Fraction 2 from the DEAE-cellulose column showed sedimenting boundaries which were a little slower than those observed in the salted out pseudoglobulin. Sedimentation-velocity coefficients for the components of the pseudo- globulin Fraction _2_, DEAE-cellulose column, indicated that the super- natant of dialyzed pseudoglobulin has only one principal component instead of the two values reported for the entire pseudoglobulin fraction. However, the precipitate of the same dialyzed pseudoglobulin Fraction 2 yielded two values. This can be interpreted as an indication that the column plays a role in the dissociation of the pseudoglobulin complex. Carbohydrate content. Smith (1946b) and Smith, Greene and Bartner (1946) presented analytical data for hexose and hexosamine. They reported higher values for hexose in pseudoglobulin, 2. 52, and lower values for hexosamine in euglobulin and pseudoglobulin; l. 58 and 1. 52, respectively. 36 SUMMARY This study was initiated for the purpose of isolating lactoglobulin fractions, separated into as many components as possible, and to determine the nature and location of the chemically bound carbohydrates. Lactoglobulins (whole lactoglobulins, i. e. pseudoglobulins and euglobu- lins) were fractionated from colostrum employing the method described E by Smith (1946b) for bovine milk. These fractions were further separated 1' into their constituent components on a DEAE-cellulose column. The J lactoglobulin fraction was separated into four components, the pseudo- Lg globulin into five, and the euglobulin into two distinct peaks and one relatively flat region consisting of many proteins having similar affinities for the adsorbent. The area measured under peak A seemed to represent proteins not adsorbed on the column. A sharp peak _B is alsolicharacter- istic for all chromatograms. Proteins comprising the areas of peaks _C_ and _D were the main components of the pseudoglobulin fraction. These two peaks in the lactoglobulin diagram seem to cover up the flat region of the euglobulin. Component _E_ of the pseudoglobulin fraction did not appear distinctly in the lactoglobulin chromatogram probably because of the rela- tively lower concentration. When pseudoglobulin Fraction 2 from the DEAE-cellulose column was dialyzed against distilled water, it separated into water insoluble and water soluble components. This suggested that one of the components plays the role of a stabilizer in the pseudoglobulin complex. It is also 37 possible that the column contributed to the denaturation of the pseudo- globulin. The water soluble component was found to be the principal component and had but one sedimenting boundary at 6. 4 Svedberg units. The analytical ultracentrifugal analysis showed that lactoglobulins contained a principal component of about 7 Svedberg units, and a second component of about 10 Svedberg units. The lactoglobulin and euglobulin fractions both showed a component of 10 Svedberg units. The lactoglob~ ulin and euglobulin fractions both showed a component of about 20. 5 Svedberg units. The pseudoglobulin fraction showed components with values of 6. 8 and 9. 7 Svedberg units. Further, all three fractions possessed one very slow sedimenting component. Carbohydrates were found to be present in all lactoglobulin frac- tions and their components. The highest carbohydrate content was in the euglobulin fraction, 9. 46 per cent. Pseudoglobulins had a total carbohydrate content of 5. 84 per cent. The concentrations of hexose, hexosamine, fucose and neuraminic acid were found highest in the euglobulin fraction. The fractions obtained from the fractionation on the DEAE-cellulose column showed a different distribution of carbo- hydrates. The concentration of hexose was the highest in the euglobulin in Fraction i--3. 90 per cent. The pseudoglobulin Fraction _1 possessed the highest values for hexosamine, fucose and neuraminic acid; 3. 15, 0. 98, and 3. 18 per cent, respectively. 38 CONCLUSIONS The fractionation of bovine milk and colostrum showed that the best method for isolating electrophoretically homogeneous lactoglobulin components was achieved through the salting out procedure with ammo- nium sulfate (Figure 1). Fractionation of the electrophoretically homogeneous lactoglobulin fractions on the DEAE-cellulose column resulted in the resolution of these fractions into several components. Thus, the lactoglobulin frac- tion was separated into four components; the pseudoglobulin into five components; the euglobulin into two distinct components and a fraction consisting of several proteins having similar affinities for the adsorbent. The main component of pseudoglobulin fraction from the DEAE- cellulose column, when dialyzed against distilled water, separated into water insoluble and water soluble components. This suggested that one of the pseudoglobulin components emerging from the DEAE column plays the role of a stabilizer for the pseudoglobulin complex, or, that the column partially denatured the pseudoglobulin complex. Studies in the analytical ultracentrifuge revealed that all lacto- globulin fractions contained several sedimenting boundaries. The pseudoglobulin fraction, when separated on the DEAE-cellulose column, showed a component, which, after dialysis, had only one sedimenting boundary. Carbohydrates were found in all lactoglobulin fractions and their components. Euglobulins had the highest carbohydrate content. 39 Pseudoglobulin Fraction 1 from the DEAE-cellulose column showed the highest concentration of hexosamine, fucose and neuraminic acid. (1) (Z) (3) (4) (5) (6) (7) (8) (9) (10) (11) 40 REFERENCES CITED Bauer, J., und Engel, St. 1911. Ueber die Chemische und Biologische Differenzierung der drei Eiweisskoerper in der Kuh- und Frauenmilch. Biochem. Zeitschrift, _3_1: 46-64. Crowther, C., and Raistrick, H. 1916. A comparative study of the proteins of the colostrum and milk of the cow and their relation to the serum proteins. Biochem. J. , _1_0: 434-452. Deutch, H. F. 1947. A study of whey proteins from the milk of various I animals. J. B101. Chem., 169: 437-448. 1 Dudley, H. W., and Woodman, H. E. 1918. The proteins of cow's colostrum. I. The relation be- tween the euglobulin and pseudoglobulin of cow's colos- trum. Biochem. J., 12, 339-350. Eugling, J. 1880. Forschungen auf dem Gebiete die Viehaltung. _2: 96. Glick, D. 1955. Methods of Biochemical Analysis. II. Inter'science Publ., Inc. , New York. Halliburton, W. D. 1890. The proteids of milk. J. Physiol. , _1: 448-463. Hammersten, O. 1883. Zur Frage, ob das Casein ein einheitlicher Stoff sei. Z. Physiol. Chem. , 1: 227-273. Hansen, R. G., Potter, R. L., and Phillips, P. H. 1947. Studies on proteins from bovine colostrum. II. Some amino acid analysis of a purified colostrum pseudo- globulin. J. Biol. Chem., _11_1_: 229-232. Hirsh, J., and Ahrens, H. E., Jr. 1958. The separation of complex lipid mixtures by the use of silicic acid chromatography. J. Biol. Chem. , 233: 311-320. Horejsi, J. , and Smetana, R. 1956. The isolation of gamma globulin from blood-serum by Rivanol. Acta Medica Scandinavica, 155, 65-70. 41 (12) Howe, P. E. 1921. The use of sodium sulfate as the globulin precipitant in the determination of proteins in blood. J. Biol. Chem. , :42: 93- 107. (13) Howe, P. E. 1922. The differential precipitation of the proteins of colos- trum and a method for the determination of the proteins in colostrum. J. Biol. Chem. , _5_2_, 51-68. (14) Jenness, R., Larson, B. L., McMeekin, T. L., Swanson, A.M., Whitnah, C. H., and Whitney, R. Mc L. 1956. Nomenclature of the proteins of bovine milk. J. Dairy Sci., 22, 536-541. (15) Kenyon, A. S., Anderson, R. K., and Jenness, R. 1959. Isolation of immune globulins from milk and colostrum with Rivanol. J. Dairy Sci., 4_2, 1233-1234. (16) Murthy, G. K., and Whitney, R. McL. 1958. A comparison of some of the chemical and physical properties of gamma-casein and immune globulins of milk. J. Dairy Sci., :1, 1-12. (17) Association of Official Agricultural Chemists 1950. Official Methods of Analysis of the Association of Official Agricultural Chemists. Assoc. of Official Agricultural Chemists, Washington, D.C. (18) Pierce, A. E. 1955. Electrophoretic and immunological studies on sera from calves from birth to weaning. 11. Electrophoretic and serological studies with special reference to the normal and induced agglutinins to trichomonas foetus. J. Hyg. . _5__3, 261-275. (19) Rackis, J. J., Sasame, H. A., Anderson, R. L., and Smith, A.K. 1959. Chromatography of soybean proteins. 1. Fractionation of whey proteins on diethylaminoethyl—cellulose. J. Am. Chem. Soc., _8_1., 6265-6270. (20) Schlossmann, A. 1896-97. Ueber die Eiweisstoffe der Milch und die Methoden .. Ihrer Trennung. Zeitsch. Physiol. Chem., _2_2_, 197-446- (21) Sebelien, J. . 1885. Beitrag zur Kenntniss (Ier Eiweisskoerper (.Ier K111111111(:I'I- Z. physiol. Chem. , 2, 445. (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) 42 Simon, G. 1901. Beitrag zur Kenntniss der Eiweisskoerper der Kuhmilch. Z. physiol. Chem., _3_3, 466-541. Smith, E. L. 1946a. The immune proteins of bovine colostrum and plasma. J. Biol. Chem. , 164, 345-358. 1 Smith, E. L. 1946b. Isolation and properties of immune lactoglobulins from bovine whey. J. Biol. Chem., 165, 665-676. Smith, E. L. 1948. The isolation and properties of immune proteins of bovine milk and colostrum and their role in immunity. J. Dairy Sci. , _3_l_, 127-138. Smith, E. L., and Brown, D. M. 1950. The sedimentation behaviour of bovine and equine immune proteins. J. Biol. Chem., 183, 241-249. Smith, E. L., and Greene, R. D. 1947. Further studies on the amino acid composition of immune proteins. J. Biol. Chem., 171, 355-362. Smith, E. L., Greene, R. D., and Bartner, E. 1946. Amino acid and carbohydrate analysis of some immune proteins. J. Biol. Chem. , 164, 359-366. Storch, K. 1897. Beitraege zur Kenntniss der Eiweisskoerper der Kuhmilch. Monatshefte fuer Chemie, _l_8_, 244-281. Woodman, H. E. 1921. A comparative investigation of the corresponding pro- teins of cow and ox serum, cow's colostrum and cow's milk by the method of protein racemisation. Biochem. J. , 2, 187-201. Yaguchi, M., Jennings, W. G., Tarassuk, N. P. 1959. Fractionation of milk proteins on anion-exchange cellulose. J. Dairy Sci., :12, 1395-1396.