FRACTIONATION AND CHARACTERIZATION OF SOLUBLE AND INSOLUBLE PROTEINS OF THE MILK FAT GLOBULE MEMBRANE By Carl Thomas Herald AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1956 A pproved 1 ABSTRACT CARL T. HERALD Isolation procedures and some- physical and chemical characteristics of the fat-globule membrane proteins were studied. Five concentrations of cold ethanol, four of cold n-butanol, and room temperature n-butanol were investigated as agents to disrupt the membrane lipoprotein complex. Membrane proteins pretreated with 35 percent ethanol (final concentration about 2o percent) were most amenable to physico-chemical studies. Cold ethanol treatment was more satisfactory than cold or room temperature n-butanol as a pretreatment for disrupting the membrane lipoprotein complex . Based upon solubility in 0.02 M sodium chloride solution, the membrane proteins were separated into soluble and In­ soluble fractions. The soluble fraction had a strong Molisch reaction, reduced Fehllng's solution subsequent to mild acid hydrolysis, was sulfhydryl negative after heating to 7 5 ° C ., and had nitrogen values varying with preparation from 9*5 to 11.5 percent. An average of seven-fold more phosphatase activity was found In the soluble than in the insoluble fraction. A single skewed component was observed in alkaline buffers when the soluble fraction was studied electrophoretically. For these components, regression equations Y = 8.840-1.762X, Y = 7 •2 5 0 - 1 .455X, and Y = 4.663 -0.900X were found which gave isoelectric points at pH 5*02, 4.98, and 5-13 respectively. CARL T. HERALD ABSTRACT On the basis of sedimentation velocity data., Insolubility in half-saturated ammonium sulfate, and an isoelectric zone near pH 5.0, the soluble proteins were tentatively classified as globulin in nature. The residual fraction was insoluble in dilute acids, bases, tions. 25 percent sulfuric acid, and 6 or 8 molar urea solu­ Sodium sulfide and sodium lauryl sulfate were r e ­ presentative solubilizing agents. Nitrogen values ranged with preparations from 1 2 .9 to 13-9 percent. A qualitative sulfhydryl-positlve reaction was obtained upon heating the protein moiety to 75° C . The insoluble fraction contained 5.6 times more iron, 25 times more molybdenum, and 10 times more xanthine oxidase activity than the soluble fraction. Electrophoretically, sodium sulfide-solubilized Insoluble protein showed one homogeneous component and detergentsolubilized protein had two prominent and one minor com­ ponents . On the basis of insolubility in the usual protein solvents, reactivity to specific reducing agents, and amino acid compo­ sition, the insoluble proteins were provisionally classified as pseudokeratin. FRACTIONATION AND CHARACTERIZATION OF SOLUBLE AND INSOLUBLE PROTEINS OF THE MILK FAT GLOBULE MEMBRANE By Carl Thomas Herald A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1956 ProQ uest Number: 10008524 All rights reserved INFO RM ATION TO ALL USERS The quality o f this reproduction is dependent upon the quality of the copy subm itted. In the unlikely event that the author did not send a com plete m anuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008524 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This w ork is protected against unauthorized copying under Title 17, United States Code M icroform Edition © ProQuest LLC. ProQ uest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 AC KN OWLEDGMENT S The author sincerely thanks Dr. J. R. Brunner, Associate Professor of Dairying, for his willing council and encourage­ ment during the investigation, and for his assistance in the preparation of this manuscript. C. W. Duncan, Professor Thanks are also extended to (Research) of Agricultural Chemistry, for critically reading the manuscript, and for arranging for some of the chemical analyses. Grateful acknowledgment is also due to Dr. R. J. Evans, Professor (Research) of Agricultural Chemistry, for his guidance and cooperation with the amino acid analyses; Miss Doris Bauer for the cystine analyses; Madden, Assistant Professor of Dairying, to to Dr. D. E. for the statistical i analyses; to S. T. Bass, Instructor (Research) of Agricul­ tural Chemistry, for the spectrographic analyses; R. M. Grimes, Assistant (Research) to Dr. Professor of Agricultural Chemistry for many fine suggestions; and, to Dr. C. A. ZIttle, Eastern Utilization Research Branch, for the enzyme analyse s . The author Is, indeed, most grateful to his wife, Norma Jean, for her assistance in preparing the manuscript, and her encouragement during the course of the study. The writer deeply appreciates the financial support of the Dairy Remembrance Fund, and the funds and facilities provided by Michigan State University and Michigan Agricul­ tural Experiment Station to make this study possible. i i TABLE OF CONTENTS 1-age INTRODUCTION ................................................... REVIEW OF LITERATURE Historical 1 . ................................................ Commentary and Objectives EXPERIMENTAL PROCEDURE 3 .............................. 10 ....................................... 12 Exploratory Investigations .............................. Concentration of the membrane material .......... Removal of lipid from the native membrane Solubility studies . . . . 12 12 12 ................................ 13 Lipid content of the lyophilized residual proteins .................................. 13 Extraction of the dry residual membrane proteins with either veronal buffer solution or w a t e r ....................................13 Procedure for the Isolation of Fat-Membrane Proteins . Preparation of the fat-globule membrane proteins .................................. Analytical Methods ............................... ... 14 14 l6 N i t r o g e n .................................... lo P h o s p h o r u s .......................................... 16 Sulfhydryl groups .................................. l6 Fat and total s o l i d s ............................... 17 Amino a c i d s .............................. Ultraviolet analyses Infrared analyses .............................. 17 .................................. 18 A s h ................................................... 18 i i i Page Solubilization of the insoluble proteins Electrophoresis . . . . 18 ................................... 18 R E S U L T S ........................................................ 23 Exploratory Investigations ............................. Effect of various alcohol pretreatments Electrophoretic characteristics 23 ......... 23 .................. 23 Results of Physical and Chemical Studies ............. 28 S o l u b i l i t y .......................................... 28 E n z y m e s ............................................... 29 Chemical composition ............................. 29 Ultraviolet analyses ............................. 29 ................................. 29 Infrared analyses Amino a c i d s .......................................... 30 Electrophoresis ................................... 30 D I S C U S S I O N ..................................................... 51 Effect of Various Procedures Used to Isolate the Membrane Proteins ................................. 51 Raw m a t e r i a l s ..................................... 51 Concentration of membrane materials ............. 52 Effect of various organic solvents ............. 53 Chemical, Physical, and Biological Characteristics of the Fat-Globule Membrane Proteins .................. 55 .................. 55 Electrophoretic characteristics Isoelectric zone for soluble proteins ........... 60 ............................... 6l ................................... 63 Solubility studies Enzyme studies Chemical composition ............................. 65 Ultraviolet analyses ............................. 68 iv Infrared a n a l y s e s ................................. 69 Ari.ino a c i d s ........................................ 69 Spectrographlc analyses SUMMARY AND CONCLUSIONS LITERATURE CITED ... 7^ ................................... 75 ............................................ 78 v LIST OF TABLES TABLE 1. 2. 3. 4. 5. 6. ?. Page SUMMARY OF THE QUANTITATIVE, UNIDIMENSIONAL ............. BUFFERED PAPER CHROMATOGRAPHY PROCEDURE 21 NITROGEN VALUES, LIPID CONTENT, AND SOLUBILITY DATA OBTAINED FOR MEMBRANE PROTEINS PRETREATED WITH VARIOUS CONCENTRATIONS OF EITHER COLD ETHANOL OR n - B U T A N O L ................................. .. 25 NITROGEN CONTENT OF MEMBRANE PROTEINS PRETREATED WITH COLD ETHANOL OR ROOM TEMPERATURE n-BUTANOL. . . . 25 SOLUBILITY CHARACTERISTICS OF THE SOLUBLE AND INSOLUBLE MEMBRANE PROTEINS ...................... 31 PHOSPHATASE AND XANTHINE OXIDASE ACTIVITY ASSOCIATED WITH SOLUBLE AND INSOLUBLE MEMBRANE PROTEINS ......... 32 CHEMICAL AND PHYSICAL PROPERTIES OF THE SOLUBLE .................. AND INSOLUBLE FAT-MEMBRANE PROTEINS 33 COMPARISON OF THE AMINO ACID COMPOSITION OF THE FRACTIONATED MEMBRANE PROTEINS WITH LITERATURE VALUES (All values c a l c u l a t e to 15 g. of' N ) 3^ ... 8. MOBILITY AND PEAK AREAS IN THE ELECTROPHORETIC PATTERNS OF THE FAT-MEMBRANE PROTEIN COMPONENTS AS SHOWN IN FIGURES II TO V I ............................. 35 9. SPECTROGRAPHIC ANALYSES OF THE ASH OF THE ............................. MEMBRANE-PROTEIN RESIDUES Vi 36 LIST OF FIGURES Pag FIGURE I. II . Ill . IV. V. VI . VII. VIII . IX . X. XI. XII. XIII. Procedure for separating the fat-globule membrane proteins ............. . ............... 22 Effect of alcohol pretreatments and solvents on the electrophoretic mobility characteristics of the soluble membrane proteins ........... . . . 2 6 , 27 Electrophoretic mobility patterns of soluble membrane proteins in various buffers ............. 37, 38 Electrophoretic mobility patterns of Insoluble membrane proteins solubilized with sodium sulfide „ * 39 Electrophoretic mobility patterns of insoluble membrane proteins solubilized with detergents , , 4° . . Electrophoretic mobility patterns of sodium sulfide-treated fractions of the fat membrane ...................... In veronal buffer at pH 8.7 41 Ultraviolet spectrogram of the soluble membrane proteins................................... 42 Ultraviolet spectrogram of the insoluble .................................. membrane proteins 43 Infrared spectrogram representative of the soluble and Insoluble membrane proteins. . . . . . 44 pH-mobility curve for the leading component of the soluble fat-membrane proteins . . . . . . . 45 Quantitative amino acid chromatogram showing: A-aspartic acid, B-glutamic acid, C-serine, D-glycine, E-threonine, and F-alanine ............... 46 Quantitative amino acid chromatogram showing: A-tyrosine, B-histidine, C-valine, and D-methionine. . 47 Quantitative amino acid chromatogram showing: A-proline. Top-portlon of chromatogram from the solvent system benzyl and n-butyl alcohols. After development with isaBln, the spots were greenish-blue against a yellow background................ 48 v ii FIGURE XIV. Quantitative amino acid chromatogram showing; A-isoleucine, and B-leucine. Lower portion of chromatogram from the solvent system benzyl and n-butyl alcohols.................. XV. Quantitative amino acid chromatogram showing; A-phenylalanine............................... INTRODUCTION Milk fat exists in milk in the form of tiny globules surrounded by a complex of proteins and lipid materials. These globules,, which average three to four microns in di­ ameter, are coarsely dispersed and in time, will rise to the top of the serum, due to a lower specific gravity, to form a cream layer. The function of the membrane surrounding the fat globules is of considerable fundamental and practical interest. In particular, the protein portion of the membrane has been a source of wide disagreement among researchers. Certain scholars of dairying chose to believe that no membrane existed, even after a membrane-like material had been demonstrated. Several attempts have been made to Identify the membrane pro­ teins with casein or whey proteins. Some have suggested that a unique protein surrounds the globules which defies existing classification. More recently, a hypothesis was presented which suggested that fat globules are surrounded by a con­ tinuous protein layer to which are adsorbed microsomes. There are data in the literature which totally or par­ tially refute the above notions concerning the membrane pro­ teins. For Instance, an electrophoretic study showed that the so-called membrane protein is heterogeneous. The origin of the membrane Is still In the stage of speculation. There are no data available showing the time at which fat globules are covered with the membrane material. 1 2 Although these considerations are in the realm of the physiol­ ogist, it would seem that a study of the membrane proteins with the currently available chemical and physical techniques should yield data suggesting a logical classification and source of these proteins. In practice, the fat-globule membrane has been shown to be intimately associated with copper-induced oxidized flavors, rancidity in milk, and with problems dealing with de-emulsification of the fat globules. Thus, it is clear that chemical and physical data related to the milk fat globule should be significant from a fundamental and practical standpoint. Some of the problems and considerations inherent to the m e m ­ brane proteins are presented in this manuscript. The development of the adapted procedure is reported under the exploratory investigation section. In the proce­ dure section are given the methods used to prepare and char­ acterize the soluble and Insoluble fractions from the mem­ brane proteins. REVIEW OF LITERATURE Historical A considerable amount of study has been devoted to the chemistry and physical phenomena associated with the "membrane" of the milk: fat globule. Recently, King (1955) has written an excellent, comprehensive review in this area of research. Ascherson (1840) was the first to postulate that a m e m ­ brane surrounded the milk fat globules. In his experiments, olive oil in contact with egg white, acquired a membrane which Ascherson called "haptogen." He reasoned that milk fat should behave in a similar manner in milk. Babcock (1889) believed that a similarity existed between the coagulation of blood and the creaming of milk and reported that fat globules were surrounded by fibrin. Hekma (1922). Storch This hypothesis was later disproved by (1897) introduced the technique of wash­ ing cream to remove materials not closely associated with the fat globules and postulated that a mucin-like protein sur­ rounded the fat globules. Abderhalden and Voltz (1909) reported that the protein portion of the membrane was not casein. Palmer and Samuelson (19^4) indicated that the emulsion-stabilizing substances adhering to the fat globules in cow's milk consisted of a single globulin-like protein and a mixture of phosphatides of undetermined nature. Employing a rather unique isolation procedure, Hattori (19^ 5 ) Isolated a proteinaceous membrane material which he 3 4 called "hapteln." Haptein was unlike any other known milk protein in respect to solubility, sulfur, and cystine content. He suggested that haptein was a keratln-like protein. Sommer and Hart Titus, (1928) concluded that the membrane protein was quite similar to casein. These workers suggested that their preparation was contaminated with some unknown substance since the isolated protein would not dissolve in 0-5 N sodium hydroxide. Rahn and Sharp (1928) believed that the foaming property of milk was due to "schaumstoff," which was thought to be a protein closely associated with the fat globules. Furthermore, soluble. schaumstoff was reported to be completely in ­ Weise and Palmer (1932) prepared a series of arti­ ficial emulsions of butterfat stabilized with either calcium caseinate, whey proteins, or lecithin prepared from egg yolks and concluded that none of the substances constituted the sole material adsorbed on the surface of the fat globules in normal cow's milk. Palmer and Weise (1933) showed that the protein of the membrane was closely associated with phosphollpldes and highmelting triglycerides. Hot ethyl alcohol and ethyl ether were used to extract the lipid materials from the membrane protein. Attempts to place the protein in solution resulted In cloudy, milky suspensions. Furthermore, perties, as well as the nitrogen, the physical pro­ sulfer, and phosphorus content of this preparation, did not correspond with similar characteristics of known milk proteins. These investigators obtained 0.66 to O .89 grams of membrane material per 100 grams of milk fat. In a later publication, Weise and Palmer 5 (193^) reported the Van Slyke nitrogen distribution for the membrane proteins. Rimpila and Palmer (1935) found that the membrane contained phosphatase which was not completely r e ­ moved after the cream had been washed several times. and Tarassuk (1936, 19^0) and Tarassuk and Palmer Palmer (1939) studied a series of artificial milks in an attempt to explain certain surface phenomena associated with the milk-fat membr a n e . According to Pedersen (1936), separator slime contained an insoluble material which he assumed to be related to casein. Jack and Dahle (1937a, 1937b) and Dahle and Jack (1937) e m ­ ployed an electrophoretic technique to study the fat globules of milk. Electrokinetic mobilities for fat globules in a c e ­ tate and citrate buffers revealed an isoelectric point at pH 4.3* Furthermore, the data suggested the probability of a double layer of phosphollpide and protein on the surface of the fat globules. Sandelln (19^1) isolated an ammonia- insoluble protein material from milk which he thought to be identical with the membrane protein. The insoluble material was isolated by mixing milk or cream with 25 percent ammonia and alcohol which was followed by centrifugation. Sandelin reported a sulfur content of 0.57 percent which is much lower than the values reported by Hattori (1925)> Weise and Palmer (193^), and Hare, Schwartz and Weese Palmer (19^*0 * in his review, (1952). postulated that the m e m ­ brane consisted of a special group of substances whose origin might be due to their greater capillary activity. and Palmer Jenness (19^5a) demonstrated that the ratio of protein to 6 phosphollplde was not identical In buttermilk and butter serum. The butter serum fraction contained a decidedly greater amount of phospholiplde per unit of protein. Mulder (19^9) believed that the surface layer must not be regarded as having been adsorbed from the milk plasma, but that the membrane consisted of components of the protoplasm of the cells of the lactiferous glands and of substances a d ­ sorbed from the milk plasma. Hare ejb al . (195^) studied the amino acid composition of the membrane materials and found more arginine, glycine and phenylalanine and less aspartic acid and leucine were in the membrane protein than in other milk proteins. Brunner, Duncan and Trout (1953a) used cold alcohol with subsequent ether extractions to purify the mem­ brane proteins. On the basis of nitrogen content, these treatments were as effective in removing lipid as that of earlier investigators who used hot organic solvents. An electrophoretic study of the membrane proteins by Brunner, Lillevik, Trout and Duncan as four components. (1953c) revealed as many The electrophoretic patterns Indicated that the protein components were of a closely associated nature. On the basis of further study with an ultracentrifuge, Brunner, Duncan, Trout and MacKenzie (1953b) reported one principal component, S20 ” 7 -3 * which suggested that the mem­ brane protein was "globulin" in nature. According to Morton (195^), milk fat globules are sur­ rounded by a continuous protein membrane on which are a d ­ sorbed microsomes. Electron microphotographs were presented to support his theory. Based on electrophoretic studies, Sasaki, Koyama and Nishikawa (1956) concluded that the fat membrane material was similar to whey proteins less betalactoglobulin. Sasaki and Koyama (1956) postulated that the fat membrane does not contain casein or beta-lactoglobulin but a part of the whey proteins plus a possible unknown or specific protein. Considerable attention has been devoted to certain sub­ stances associated with the membrane proteins. Certain pro­ teins in equilibrium between the membrane-plasma interface as well as specific compounds of structural significance have been studied. Sharp (19^0) estimated that the fat glob­ ules in a quart of Guernsey milk had a total surface area of about 1050 square feet. Trout, Halloran and Gould (1935) showed that homogenization increased the surface area of fat globules about five or six times. Trout (1950) presented additional information concerning the role of surface area in normal and homogenized milk. The fact that cream which was separated from cold milk contained an agglutinating material not commonly found in the cream separated from warm milk was observed by Sharp and Krukovsky (1939)* This material, capable of causing fat clustering, was isolated and identified as euglobulin by Dunkley and Sommer (19^*0 • Peters and Trout (19^5) reported that an attraction, based in part upon oppositely charged particles, existed between the fat globules and the leucocytes The positively charged leucocytes were attracted the most at pH values near the Isoelectric point of milk casein and the least at lower pH values. Sommer (1951) postulated that fat 8 globules exist in milk plasma surrounded by an atmosphere of reversibly adsorbed materials, "principally ions." The en ­ zyme phosphatase, which is associated with the membrane, has been studied by Kay and Graham (1933), Morton Zittle, DellaMonica, Custer and Rudd and Zittle and DellaMonica (195*0* and (1956b). Morton (1950) (1952) reported that n-butanol had the specific ability to induce the separation of lipo­ protein complexes. Morton (1950) treated buttermilk from washed cream with n-butanol and obtained a 5000-fold increase in the concentration of phosphatase, as compared with milk. Other materials reported to be associated with the membrane include xanthine oxidase, diaphorase, DPN-cytochrome c reductase and approximately 11 percent nucleic acid 195*0 . (Morton, Zittle et_ a l . (1956b) employed similar analytical methods and found less than 0.5 percent nucleic acid associ­ ated with the membrane. Further, they theorized that micro- somes contained protein, phospholipide, and nucleic acid which might be pictured as a parcel of enzymes cemented together with phospholipides and nucleic acid. Toyama (1932), Ball (1949), Morton (1939), Polonovskl, Baudu and Neuzil (195*0, Avis, Bergel and Bray (1955) and Zittle et al^ (1956b) have shown that the fat globule me m ­ brane is rich in xanthine oxidase. The association of iron and copper with the fat-globule membrane was shown by Davies (1933)• Kon, Mawson and Thompson (l9*+*0 stated that carot- enoids and cholesterol were present in large amounts when the ratio of the fat-globule surface to fat was high. Eaton and Patton White, (195*+) assumed that carotenoids were in a 9 dilute solution or in a loose chemical complex on the globule surface. The carotenoid concentration in the phospholipide membrane was calculated to be 0.0645 percent as compared to 0.000476 percent in the interior of the fat globule. Palmer and Weise (1933) demonstrated that phosphollpldes were associated with the fat membrane. Kurtz, Jamieson and Holm (193*0 isolated an ether-soluble lecithin-cephalin fraction (about 56 percent lecithin and *+4 percent cephalln) which contained 70.6 percent oleic acid. The role of phospho- lipides in dairy products was demonstrated by Thurston, Brown and Dustman (1936). These investigators showed that phospholipides were strong contributors to the so-called "rich flavor" of milk. Moreover, the oxidized flavor called "oily-stale” was shown to be intimately associated with phospholipides. A technique for the preparation of phospho- llpides developed by Olson (1944) yielded one gram of phos- pholipide from about 1500 pounds of milk. Jenness and Palmer (1945b) reported a high-melting triglyceride in close associ­ ation with the fat membrane. Dahle and Pyenson (1938), Gould and Sommer Townley and Gould (1943) have sulfhydryl groups demonstrated the from heated to Townley and Gould (1939)* and presence of membrane proteins. According (1943)> heat labile sulfides of milk originate from two sources: first, the milk serum, and second, the material associated with and firmly attached to the fat globules. washing procedure The effect of heating cream prior to the upon the subsequent recovery of membrane protein was demonstrated by Loewenstein and Gould (1954). 10 . Milk which was heated to 40°C . momentarily, 6 2 ° C . for 30 minutes, and 82° C . for 15 minutes yielded membrane material that contained 2 1 .8 6 , 1 5 -5^> &nd 7*70 percent protein, res pectlvely. Commentary and Objectives The literature yields little specific information which would permit a logical classification of the membrane proteins, but theories concerning the nature and origin of the membrane constituents are abundant. Unfortunately, some of these theories are based on a few or no laboratory observations. The complexity of the membrane has undoubtedly encouraged some workers to seek areas of more lucrative data. Recently published reports propose that the protein portion of the fat membrane is of cellular origin, which excludes casein and the whey proteins. The application of electrophoresis and ultracentrifugal techniques to the study represent the best approach available at this time. A vexing problem confronting investigators Interested In this problem has been the insolubility of a fraction of the membrane. Obviously, the techniques mentioned previously are of limited value unless the materials in solution are representative samples. The primary objective of this investigation was to devise a technique which would render the membrane proteins soluble. A minimum of change was desired for proteins to be used in physico-chemical studies. Subsequent to this acc o m ­ plishment it was hoped to characterize the membrane proteins by several techniques. Thus, with more data available, 11 certain clues might be obtained which would reveal a better insight as to the origin, role and nature of the fat-globule membrane proteins. EXPERIMENTAL PROCEDURE The milk used throughout this study was from a mixed herd source. Fresh, raw milk of about 3*5 percent fat and 12.3 percent total solids was obtained from the Michigan State University Creamery. Exploratory Investigations There is paucity of information in the literature on methods to render the membrane proteins soluble. Thus, a series of experiments were planned in which existing methods were reviewed and new Ideas investigated. buttermilk Washed-cream (hereafter referred to as serum) was obtained from washed cream prepared according to classical methods (Storch, 1897; Weise and Palmer, 193^; and Brunner et a l ., 1953a). Concentration of the membrane material. Pour methods based on different physical and chemical principles were investigated: l) The condensing procedure of Brunner e_t al . (1953a), 2) Isoelectric precipitation at pH 3-9> according to Palmer and Weise (1933)? 3) Freezing the serum slowly to concentrate the membrane materials, and 4) Salting-out the proteins from the serum with 50 percent ammonium sulfate. Removal of lipid from the native membrane. Concentrated membrane materials were treated with varied concentrations of cold (0° to ~5°C.) ethanol or n-butanol. Alcohols were removed by washing the cold mixture several times with cold 12 13 ethyl ether on No. 192 Eaton and Dikeman folded filter paper. Several extractions with 3 0°c. ether were required to remove most of the lipid materials. The residual proteins were dia- lyzed, lyophilized, and stored in the dry state at -20° C . The intermittent freezing and thawing method of McFarlane (19^2 ) was also investigated. Solubility studies ^ One-tenth gram of dry protein was added to phosphate buffer of pH 7-o. The buffer was composed of 0.02 M phosphate and 0.15 M sodium chloride. ing for 2k After stand­ hours at 5°C ., these solutions were centrifuged for 40 minutes at 12,800 G in a Model SS-1 Servall Centrifuge. Protein solubility was determined as soluble protein nitrogen according to the modified Kjeldahl method of Menefree and Overman (19*10) . The results were expressed as milligrams percent of nitrogen. Lipid content of the lyophilized residual proteins. modified procedure of Mojonnier and Troy this purpose. A (1925) was used for Fifty milligrams of membrane protein were weighed into a 15 milliliter centrifuge tube and treated with 1 milliliter hot water, a few drops ammonium hydroxide, 0.2 milliliter ethanol, and 5 milliliters ethyl ether. The mixture was agitated and the supernatant was removed after centrifuging for approximately two minutes. The residue was extracted twice with ethyl ether before drying and rewelghing. Loss In weight was considered to be lipid material. Extraction of the dry residual membrane proteins with either veronal buffer solution or water. Lipid-extracted, 14 lyophllized membrane proteins were dispersed in either veronal buffer solution or tap water. The soluble materials taken up in the aqueous phase were separated frorn the mixture by centri­ fugation at 25,000 G for 30 minutes. The supernatant was lyophllized and dissolved in selected buffer systems for standard electrophoretic analysis. Procedure for the Isolation of Fat-Membrane Proteins The methods selected for the isolation and character­ ization of the membrane proteins were based on the experi­ ences of other researchers and our own modifications. Sim­ plicity, the element of time, and possible undesirable changes in the protein system were prime considerations in the devel­ opment of the isolation procedure. Preparatlon of the fat-globule membrane proteins. The fat-globule membrane proteins were prepared according to the flow diagram shown in Figure I. was originated by Storch Palmer (1932). The cream-washing procedure (1897) and refined by Weise and Brunner et_ al . (1953a) introduced the concept of cold ethanol treatment as a pretreatment for lipid r e ­ moval . Concentration of the membrane material from serum by salting-out with ammonium sulfate and subsequent frac­ tionation based on solubility in 0.02 M sodium chloride were adapted from exploratory studies. Thirty percent cream was prepared from fresh, raw milk separated at 46°C . with a Model 518 DeLaval Laboratory Sepa­ rator. The cream was washed six times with three volumes of 4 0 ° C . tap water which produced a washed cream of about 55 15 percent fat. After standing overnight at 5°C ., the washed cream was churned in a glass jar agitated horizontally with a mechanical device. Butterfat was removed by warming the churned mixture to about 4 0 ° C . and separating with a DeLaval laboratory separator. The fat-membrane material was salted out of the serum at room temperature by adding slowly with agitation a saturated solution of ammonium sulfate until a final concentration of 55 percent ammonium sulfate was reached. Concentration of the membrane material was achieved by cen­ trifuging at 25,000 G for 30 minutes in a Model SS-1 Servall Centrifuge. Fifty grams of protein-containing material were treated with 100 milliliters of 35 percent ethanol in ether which resulted in a final ethanol concentration of about 26 percent. The temperature was held at 0° to -5°C . while the mixture was agitated for ten minutes. Alcohol was r e ­ moved by filtration in a refrigerated room (-20°C.) followed by five washings with 200 milliliters of cold ether. Finally the material was extracted three times with 250 milliliters of ethyl ether at 3 0 ° C . The proteins were made into a thick slurry with 0.02 M sodium chloride solution and held over­ night under 29 inches of vacuum to remove residual ether. The resulting protein concentrate was separated into soluble and insoluble fraction by extracting with four 150 milli­ liters of 0.02 M sodium chloride solution and subsequent cen­ trifugation at 25,000 G for 30 minutes. The supernatant con­ tained the "soluble” fraction, whereas the pellet in the bottom of the centriguge tube was designated as the "insol­ uble" fraction. The protein solutions were dialyzed further 16 against 0.02 M sodium chloride solution and stored at 5°C . Analytical Methods Nitrogen. The method of Menefree and Overman was modified for this purpose. (l9;+0) A fifty milligram sample of protein was digested slowly for about two hours with ten milli­ liters (N-free) sulfuric acid, O.lA grams of mercuric oxide, and two grams of sodium sulfate. Following digestion 70 milli­ liters of distilled water and 30 milliliters of 50 percent so­ dium hydroxide containing sodium thlosulfate were a d d e d . The mixture was steam distilled for ten minutes into 25 milliliters of boric acid solution containing four drops of indicator. This was titrated with approximately 0.05 N sulfuric acid from a burette calibrated to 0.05 milliliters. Phosphorus. The protein samples were digested by modi­ fying the procedure of Horecker, Ma and Haas method of FIske and Subbarow opment. (19^0). The (19^5) was used for color devel­ Fifty milligrams of the protein preparation were digested to a brown color with five milliliters of 10 N sulfuric acid after which five drops of 30 percent hydrogen peroxide were added and the solution heated to clearness. A standard curve was prepared by plotting optical density against known phosphorus concentrations. dilutions and color development, After appropriate the phosphorus content of the unknown samples was read directly from a prepared stand­ ard curve . Sulfhydryl groups. The method of Josephson and Doan (1939) as modified by Patton and Josephson for this purpose. (19^9) was used 17 Fat and total solids. Total solids and fat were deter­ mined on milk, cream, and washed-cream buttermilk by the method of Kojonnier and Troy Amino acids. (1925). The unidimensional, buffered paper chroma­ tographic technique of McFarren (1951) and McFarren and Mills (1952) was adapted for this purpose. The conditions for the amino acid procedure are summarized in Table 1. The chroma­ tograms were irrigated in a Chromatocab, Model A300 Equipment Corporation) (Research on Whatman No. 1 filter paper. The standard solutions contained 0.4 microgram amino acid per microliter, except for tyrosine, which contained 0,3 micro gram per microliter. The known and unknown solutions were delivered on the chromatographic sheet in 5 microliter por­ tions. Four concentrations of the standard solution 15* and 20 microliters) were applied. (5, 10, A standard curve was prepared for each amino acid and chromatogram. The borate buffer was modified to exclude potassium chloride and the phosphate buffer was modified to 0.02 M for the argininelysine run. After the color developed, the spots were cut out and eluted with 5 milliliters of a 1:1 mixture of iso­ propyl alcohol and water. Optical density was determined at 570 millimicrons in a Beckman Model B Spectrophotometer. Standard curves were prepared by plotting the concentration of the amino acid against the optical density. Amino acid concentrations in the unknown samples were read directly from the appropriate standard curve. Ultraviolet analyses. A Beckman Model DK 2 Spectrophoto­ meter was used to scan the protein solutions over the spectrum 18 range of 220 to 340 millimicrons. Protein concentration was 0.02 percent in 0.02 M sodium chloride solution. Infrared analyses. Nujol mulls were scanned between salt plates from 2 to 15 microns with a Perkin-Elmer Model 21 Spectrophotometer. Ash. Two-gram samples were placed in a muffle furnace at 550° C. for 12 hours. Solubilization of the Insoluble protei n s . The protein materials which were not soluble In ordinary protein solvents were treated with the following types of reagents: dilute acids and alkali, strong acids and alkali, urea, sodium sul­ fide and thioglycollc acid, sodium lauryl sulfate, and dodecyl benzene sodium sulfonate. The insoluble proteins were sus­ pended in 0.02 M sodium chloride solution in these experiments. Electrophoresis. Electrophoretic mobility patterns were recorded at approximately 1 ° G . with a Perkin-Elmer Model 38-A Electrophoresis Apparatus originally described by Moore and White (1948) and subsequently equipped with a Philpot- Svennson cylindrical lens system as described by Longsworth (1946) and Bull (1951). Buffers were calculated with the Henderson-Hasselbach and Ionic strength equations. cases, an ionic strength of 0.1 was used. taken from Kolthoff and Laitinen In all The pK values were (1948) and Longsworth (1952). The pH measurements on the buffers were taken with a Beckman Model G Potentio raster at 10, 15, 20, and 25°C . were plotted as Temperatures abscissas and pH values as ordinates, and pH values for 1 ° C . were obtained by extrapolation. One-half percent protein concentration was obtained by diluting the 19 original extracts with 0.02 M sodium chloride solution. Protein solutions were dialyzed for at least 24 hours at 5 ° C . against three 350 milliliter buffer changes. stirrer A mechanical (American Instrument Company) was used to facilitate dialysis. The photographic negatives of the electrophoretic images were placed In a photographic enlarger and the twice magnified image was traced on good quality graph paper. The distance from the initial boundary to the central area of each peak was measured for mobility calculations. Average values of the ascending and descending patterns were taken. To obtain precise mobilities, ary should be known. the conductivity at each bound­ When this is impossible, best results are obtained by averaging ascending and descending values (Moore, 1949)• Potential gradient, commonly referred to as field strength, was calculated as follows: F = i/aK where: F = potential gradient, I = current in amperes, a =* cross-sectional area of the U cell, and K = the specific conductivity of the buffer protein solution . Electrophoretic mobility following formula: (u) was calculated with the u (cm.^, volt-1, sec.-1) = wh e r e ; d = distance boundary traveled, a = cross-sectional area of U cell, dak tirm 20 k = conductivity cell constant, t = time in seconds, i = current in amperes, r = resistance of buffer in ohms, and m = magnification factor of optical system. Compositional distribution was determined by dropping ordinates from the lowest point between peaks and subsequently weighing the respective cut-out areas. Average values of the ascending and descending pattern area were recorded. 21 1 *-* CHROMATOGRAPHY PROCEDURE O G •H G 0 - 0 • iH *fH 4-> O Cd G 4H cd CO cd Td .— O X '•— ' OJ G «H ( D P O G O 44> G *tH G CD OJ C\J -= j-' OJ o G r | G g •H 0 cd O -=J" 50 G *H • CD £ c •H X s *»H G 4-> Q o on vo 00 OJ o -=J" o -=t- o o o o VO 00 £ ' " PAPER 50 G •rH g G O CD e *H X -— -• • G x : ■=J- -=J~ OJ OJ UNIDIMENSIONAL, BUFFERED 03 'O T3 o o CO T3 *H O cd XJ CD c *r-t o •rH £ 40 0 G C •H CD 4 -5 CJ < T5 £ cd CD cd CD o *H B CD -H O 3 G >> CO 1— I CD 1— I Xj 1— I G < O CO O X> 4 -> O *h iH X G CO 1—I >i 40 *H Cd CD X> S £* 10o o CD CD C G •rH G *rH • H CO XI O o o 50 G cd 1— 1 >1 G CD X oh 0o o X - G vo VO OJ v o o -= f OJ OJ OJ 00 VO v o 1— t 0 OF THE X CL o o G •H —\ 1 0 G Oh Xo o VO 00 O I—1 0 4-> G CD > *H 0 00 o 3 G CD • H r— 1 O 3 O CO -« rH x: o O G O o d -h CD G SUMMARY 0) G cd rH •rH cd QUANTITATIVE, on CL o in O in 00 vm mo VO 1 1 vo vo in 1 T3 x » x: >> Si a> p p in on o C Cl « 5 a> x: x: p p W II. pre-treatment P .-IO c P>»cUtrJ x: c«si FIGURE ASCENDING o\ VO the electrophoretic DESCENDING I The effect of alcohol pre-treatments and solvents characteristics of the soluble membrane proteins. I mobility 26 pre-treatment ASCENDING FIGURE II (Continued) The effect of alcohol pre-treatments and mobility characteristics of the soluble membrane proteins. Alcohol solvents on the electrophoreti DESCENDING 27 28 Results of Physical and Chemical Studies Exploratory results Indicated that the proteins of the fat-globule membrane could be placed into solubility cate­ gories. The portion soluble in the supernatant of 0.02 M sodium chloride solution after centrifugation was called the soluble fraction,, whereas the residue was designated as the insoluble fraction. The ratio of the soluble fraction to the insoluble fraction was approximately 44 percent and 56 percent, respectively. The data in the following section were used to characterize the soluble and Insoluble fractions. Solubility. The data in Table 4 Illustrate the solu­ bility characteristics of the membrane proteins. The soluble fraction was readily soluble in aqueous solvents but was readily salted-out by half saturation with ammonium sulfate. The Insoluble material resisted solution in water, organic solvents, and alkaline buffers. hydroxide, Twenty-five percent sodium sodium sulfide in a sulfide/protein ratio of 0.6 : 1 , sodium lauryl sulfate in a detergent/protein ratio of 0.8 : 1 , and dodecyl benzene sodium sulfonate in a deter­ gent/protein ratio of 1.2 : 1 were capable of effecting solution. Five milliliters of a 0.5 percent suspension of insoluble protein required 0.08 milliliters Tergitol J or 1.1 milliliters of Triton X-100 and momentary heat to 7 5 ° C . for solution. Solutions prepared with Tergitol 7 became turbid when cooled. Conversely, protein solutions which con­ tained Triton X-100 remained turbid until cooled to 62°C . and heating the mixture induced turbidity. Protein solutions 29 prepared with Triton X-100 were quite viscous. Enzymes. The data in Table 5 show the distribution of xanthine oxidase and alkaline phosphatase in the soluble and insoluble protein fraction of the membrane protein. Two samples prepared about one month apart were submitted for analyses. Enzyme activity was employed to indicate any un­ desirable changes that might occur in the protein system due to preparation technique. Xanthine oxidase was found to be concentrated in the insoluble fraction and phosphatase in the soluble portion. Chemical composition. The data in Table 6 emphasize further the differences in chemical composition between the two fractions. more ash, The soluble fraction contained significantly phosphorus, magnesium, and calcium. The insoluble fraction contained a greater quantity of nitrogen and was sulfhydryl-positive after flash heating to 75°C * Ultraviolet analyses. Absorption peaks in both fractions were most intense at 278 millimicrons. This is illustrated in Figures VII and VIII. Infrared analy s e s . A spectrum representative of the soluble and insoluble fractions is shown in Figure IX. Ab­ sorptions attributable to the functional groups in the pro­ teins were observed at 3-05, 6.05* and 9-50 microns. The spectrum of the soluble and insoluble fractions were almost fingerprint images, but the soluble portion revealed a small shoulder at 5-75 microns which was not evident In the In­ soluble portion. 30 Amino acids. The amino acid composition of the soluble and insoluble fraction is reported in Table 7* More valine, arginine, and methionine were found in the insoluble fraction than In the soluble fraction. Photographic reproductions of the developed chromatograms are presented In Figures XI to XV. Electrophoresis. Electrophoretic patterns for the sol­ uble fraction in acid and alkaline buffers are presented in Figure III. Various degrees of resolution were observed on either side of the isoelectric zone. On the acid side, two and three components were observed, but on the alkaline side, the components appeared more closely associated. In Figure X, electrophoretic mobility was plotted as the ordinate and pH as the abscissa. The intercept of the regression curve with zero mobility established the isoelec­ tric zone for the major components at about pH 5*0. Figures IV and V represent electrophoretic mobility patterns of the insoluble protein material treated with var­ ious solubilizing agents. Sodium sulfide-treated insoluble membrane proteins migrated as a single homogeneous peak. Insoluble membrane proteins treated with selected detergents migrated chiefly as closely associated two-component systems. A heat-labile third component was observed following the major components. All solubilized insoluble membrane-proteins were examined in alkaline buffers to the Isoelectric zone. 31 TABLE 4 SOLUBILITY CHARACTERISTICS OF THE SOLUBLE AND INSOLUBLE MEMBRANE PROTEINS Solubilizing Agent Water Sulfuric acid (25$) Sodium hydroxide Urea Soluble Insoluble* + - + - (25$) (6 and 8 M) + + - Sodium lauryl sulfate + Dodecyl benzene sodium sulfonate 4~ Thioglycolic acid + Sodium sulfide + + Tergitol 7 + Triton X-100 + * Material was suspended in 0.02 M sodium chloride TABLE 5 PHOSPHATASE AND XANTHINE OXIDASE ACTIVITY ASSOCIATED WITH SOLUBLE AND INSOLUBLE MEMBRANE PROTEINS Xanthine oxidase Sample3, 1 Soluble Insoluble Sample 2 Sample 1 Units/mg. protein^ *.3 42 Membrane protein0 Phosphatase 2.9 30 Sample 2 58 171 11 19 40 9 Units/ml. 143 400 Skimmilk0 23 88 VJhey0 18 28 Cream (25^ fa t )0 a The data for samples 1 and 2 were supplied in a personal communication by Dr. C. A. Zittle. b Zittle et a i . units (1956b) c These values were reported by Zittle et al_^ (1956b) 33 TABLE 6 CHEMICAL AND PHYSICAL PROPERTIES OF THE SOLUBLE AND INSOLUBLE FAT-MEMBRANE PROTEINS Soluble Sulfur o CO (%) i —1 Ash (%) 11 .10 cn {%) Phosphorus 0 .94 0.70 {%) Nitrogen Insoluble 0 .45 0.23 7.06 2 .08 - Reduce Fehling's solution Molish test + + N1troprusslde test - +a Color Biuret test a Heated momentarily to 75° C . White Brown + + 34 TABLE 7 COMPARISON OF THE AMINO ACID COMPOSITION OF THE FRACTIONATED MEMBRANE PROTEINS WITH LITERATURE VALUES (All values calculated to l6 g. of N) Fat-membrane Protein Constituent Soluble Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Serine Threonine Tryptophan Tyrosine Valine 2.35 5.48 5.41 2.65 7.83 2.34 3.90 4.35 6.65 Insoluble Total Membrane MateriaV A B C 3.82 3.17 8.22 7.60 2 .25 7.02 6 .63 10.79 3.88 4.05 4.58 8.20 7.33 1.4l 3.86 4.49 2.43 4.64 8.60 2 .96 5.31 5.31 3.60 6.70 1.88 4 .40 2.43 9.49 3.20 3.98 4.48 7.52 8.02 2.28 4.68 4.95 2.12 4.50 5.42 6.99 1 .50 12 .85 3.76 3.03 5.67 8.71 5.92 2 .08 6 .03 1.72 5 .65 5.0 1.5 3.0 1.7 3.5 9.0 6.1 2.0 6.4 0.9 5.4 a Legend: A = Values obtained when soluble fraction equals 44 and insoluble fraction equals 56 percent; B = Values reported by Brunner et a l . (1953a); and C = Values reported by Hare e_t aT.' (1952) 35 TAELE 8 MOBILITY AND PEAK AREAS IN ELECTROPHORETIC PATTERNS OF THE FAT-MEMBRANE COMPONENTS AS SHOWN IN FIGURES II TO VI ctr°ph°retlc mobility patterns -Figure Row II 1 2 3 4 5 6 III IV V VI l 2 3 4 5 O 7 8 1 2 3 A 1 A.32 4.93 5 .AA 6 .65 A.A8 A.20 5-50 2.35 3 .86 1 .Al 0.95 3.21 4.54 5.22 Mobility Peak No. 2 Area in percent 3 8 9 .6 2 .30 3 .60 90.1 88.3 91.6 90.9 100.0 3.39 4.55 2.53 A .l6 1.78 3.22 0.99 0.54 2.30 A. 05 A. 52 2.07 1.47 3.09 0.66 1 .21 2 .81 Ao.A 19-5 6.0 14.A 48.0 11.0 52.5 38.2 Peak No. 2 3 10.A 9.9 11.7 8.A 9.1 48.9 15.2 20.8 28.3 51.7 63 .8 47.5 55-9 10.7 6 5 .9 75.2 57.3 25.2 6,0 100.0 3.88 6.81 1 6.11 9.15 6.01 5.75 7.12 1 2 3 A 9.80 9.52 11.53 10.20 9-33 7.85 9.05 8.82 1 2 3 6 .19 5.5A 5.85 A. 93 5.22 5.41 3.5 16.1 8.1 8.10 6.79 6.87 32.5 3 2 .A 11.8 Al . 7 6.9 36.5 lA.l 96.5 83.9 91.9 52.9 1A.6 48.7 4.6 9.6 67.6 83.6 93.1 63.5 85.9 36 TABLE 9 SPECTOGRAFHIC ANALYSIS OF THE ASH OF THE MEMBRANE 9R0TEINS Element Soluble Aluminum Calcium Copper Iron Trace 0.603 {%) (p .p . m .) (p .p .m .) Magnesium (f6) Manganese (p.p.m.) Insoluble Trace 0.319 132 82 91 510 0.548 0.227 11 11 125 Molybdenum (p.p.m.) 5 Phosphorus (°/o) 0.4l6 0.289 Silver Trace Trac e Zinc 17 22 (p .p . m .) 37 ASCENDING DESCENDING Glycine-HCl; pH 2.05 • -► > 2 8030 sec.; 6.5^ V. c m . " 1 Citrate: • / 9235 sec.; pH 3-35 11.78 V. cm.-l Glycine-HCl; pH 3*78 9171 s e c .; 9-71 V. cm. Acetate; -- 1 -1 pH ^.30 2 3 12900 sec.; 10.36 V. c m . -1 FIGURE III. Electrophoretic mobility patterns of soluble membrane proteins in various buffers. ASCENDING DESCENDING / Acetate; pH 5*30 10525 sec.; 10.45 V. cm."l Phosphate; pH 6.10 11265 sec.; 7*21 V. Phosphate; 6300 sec.; cm."^ pH 7.30 I <---1 Z 10.74 V. c m . ”l / Veronal; 6550 sec.; Z Z pH 8.70 8.79 V. c m . “^ FIGURE III. (Continued) Electrophoretic m o b i l i t y patterns soluble membrane proteins In various buffers. 39 ASCENDING DES C E N D I N G / Phosphate; *--- ^ 6050 sec.; 6.8l V. c m . ”*; conc., Phosphate; 5010 sec.; pH 6.10 0.30$ pH 7-30 10.80 V. c m . ”*; conc., 0.40$ * 0.58$ ^ --- 1 / Veronal; 5357 sec.; 12.00 V. c m . ”*; cone., Veronal; •--- » 3410 sec.; (Sodium lauryl pH 8.70 pH 8.70 -1 11.97 V. era." ; conc., 0.50^> 1 sulfate followed by sodium sulfide) FIGURE IV. Electrophoretic mobility patterns of Insoluble membrane proteins solubilized with sodium sulfide. 40 ASCENDING Veronal; DESCENDING pH 8.70 p / 3350 sec.; 1 11.39 V. c m . " 1 ; conc., Sodium lauryl sulfate Veronal; Vei pH 8.70 0.39^ < --- * p i ^ 3370 sec.; 12.24 V. cm. 1 ; conc., 0. 5C$ 3745 sec.; 8.47 V. cm. 1 ; conc., Sodium lauryl sulfate 0.80^ < --- 1 Veronal; V e r o n a l ; pH 8.70 / ? 3130 sec.; 11.40 V. cm.~l; conc., 0.4456 Dodecyl benzene sodium sulfonate < --- • FIGURE V. Electrophoretic mobility patterns of Insoluble m embrane proteins solubilized with detergents. i o s n O 5 3 W O CO W a i i 8 OO OO m CO 00 >R > in C 5 53 K H Q s of NagS-treated fractions of the LT\ fat-membrane In 4l £ (d TO C L a> c cd to P O J ,o cd e z to E X i d> 4-> *rH d5 < o r-l - H U o to to O X3> O *T0 O H (flTJ O P« *o co 4-i (d 1 t 1 CL in in C V J 4-i O E T3 T OC O N C m «3h •>-1*H f—H 4) < d ' O *r4 o •H -O 4 >C O X> O *H 3 P. 4->T3,O-C Q. C C 0TO (d cd 0 i-H 3 C TrTHO to • o 3 *- e x: T O4-3 E -H I s +3 cd to 4h C TO TO*0 (—l4-> TO 0*0 X0 P iTJ 3 Pi*cd mobility CO FIGURE VI. Electrophoretic veronal buffer at pH 8.70 o patterns Cd FIGURE VII. Ultraviolet spectrogram of the soluble membrane proteins. 42 3 ON V l l M S N VU1 I N 3 0 i/3 d 43 OQ G •H (1) •P O Q. G u cd U *6 .G o B cd bO o u -p o cd U P W g C3 M fo 30NV±HH$NVti± IMJDdJd FIGURE IX. Infrared membrane spectrogram proteins. £ representative of the soluble and insoluble 44 (A U S N JO l V D U d O ) 3 D N V 9 V Q S 9 V 45 Mobility (m)*(cm.1,voitH,mc.h,X10**) • ■ Triol I O ■ Triol 2 □ * Y>8.840-1.762 X -7, wTrrTHTT X ^xuun pH-mobllity curve for the leading component ^ the soluble fat-membrane proteins. 46 1 ^ ^ J 1 w . 1 ' > * « » g c • *— * * * * ^ J < 1 ; V s < * * • # H • f • • • # # § f • * • 1 • # t i l l U1 i f# 0 * 0 * # # # > .j > ~« s ^ f 0 0 9 i 0 • I B C 9 D M M i i l f t • • ■ 11 I It I FIGURE XI. I Quantitative amino acid chromatogram showing: A-aspartic acid* B-glutamic acid, C-serine, D-glycine, E-threonine, and F-aianine. 4 IMP FIGURE XII. Quantitative amino acid chromatogram showing A-tyrosine, B-histidine, C-valine, and D-methionine. FIGURE XIII. Quantitative amino acid chromatogram showing; A-prollne. Top-portion of chromatogram from the solvent system benzyl and n-butyl alcohols. After development with i&atin the spots were greenish-blue against a yellow background. FIGURE XIV. Quantitative amino acid chromatogram showing; A-isoleucine, and B-leucine. Lower portion of chromatogram from the solvent system benzyl and n-butyl alcohols. 50 i i f M = 1 ! | [ n i i i * i J t t t t l t t !Tlltttlt T • • t I *# # # * *9f e#f Ht A » *■ • % FIGURE XV- Quantitative amino acid chromatogram showing: A-phenylalanine. DISCUSSION "Lipoprotein” has frequently been used to describe the fat-globule membrane complex. Since the membrane is composed of protein, high-melting triglycerides, and phospholipides, this term has justification. cussion, Hence, in the following dis­ lipoprotein will be used synonomously with the fat- globule membrane. The isolated lipoprotein in this study does not represent the Intact membrane; however, the isolated materials represent the membrane most closely associated with the fat-globules. Effect of Various Procedures Used to Isolate the Membrane Proteins Raw materials. stored Attempts to wash cream which had been (for about 24 hours) at 5°C . were unsuccessful. Aged cream "oiled-off" rapidly after two or three washings and in some cases completely de-emulsified. Cream prepared from fresh, raw milk did not oil-off during the washing procedure consisting of six washings. Jenness and Palmer (I9^5a) emphasized the Importance of using fresh milk in preparing washed cream. These investigators isolated 0.66 grams protein and 10.8 milligrams of phosphorus per 100 grams of fat from washed cream prepared from uncooled raw milk. Washed cream prepared from milk aged for 24 hours at 3 .3°C. only yielded 0.42 grams of protein and 9*7 milligrams of phosphorus per 100 grams of fat. These data can be Interpreted to show that 51 52 a portion of the phosphorus was concentrated closer to the fat globules than to the proteins. The phospholipides of the membrane would be a logical source of phosphorus. Viscous, stable emulsions frequently formed whenever washed cream was churned in a conventional laboratory churn. Washed cream was de-emulsified most rapidly in a horizontally agitated gallon jar. Thirty gallons of milk yielded 4,ll6 grams butterfat, and ^,306 grams of serum of pH 7*0 containing 1.22 percent and 0.78 percent total solids and fat, respectively. Thus, I .27 grams of membrane material per 100 grams of butterfat were recovered. Since the membrane was found to contain about 40 percent protein, the recovery of protein can be estimated at about 0.51 grams per 100 grams of fat. and Palmer Jenness (l9^5a) reported a protein recovery ranging from 0.^6 to 0.86 grams per 100 grams of fat. Concentration of membrane materials. Approximately 16 to 20 hours were required to condense serum according to the method of Brunner ejb al . (1953a) . procedure, During the condensing the serum foamed considerably and a large portion of membrane material adhered to the condensing flask. ing to Neurath and Bull Accord­ (1938), surface-film formation and adsorption at interfaces were conducive to the denaturation of proteins. Moreover, the condensed mixture froze when cooled to about 0° C ., and cold ethanol combined with the material to form an unwieldy emulsion. Slowly freezing the serum to concentrate the membrane material did not concentrate the solids in a satisfactory manner. Isoelectric-precipitated membrane material subse­ quently pretreated with ethanol and extracted with ether resisted solution. According to Lisse (1940), proteins at the isoelectric point are most sensitive to alcohol. Further, the supernatant from the isoelectric-precipitated material contained a yellowish-green fluorescent material which suggests that a form of riboflavin might be associated with the mem­ brane proteins. Ball (1939) reported that flavin adenine dinucleotide was closely associated with membrane proteins. Membrane material was readily concentrated from 32 to 34 percent solids upon adjusting the serum to 55 percent ammonium sulfate concentration and subsequent centrifugation. The supernatant was biuret-negative indicating a complete removal of proteins. Membrane material was not removed efficiently from the serum whenever a 50 percent ammonium sulfate concentrate was used. The concentrated membrane material had a reddish-brown color. When the ammonium sulfate precipitate was held at 0 to -5°C . and treated with cold ethanol and ether, no hard masses or emulsions were formed. Alcohol was readily removed from the mixture when it was fil­ tered and washed several times with cold ether in a -20° C . cold room. The residue on the filter paper had a friable appearance following the cold ether extractions. Effect of various organic solvents. The data in Table 2 show that membrane proteins pretreated with 35 percent ethanol had the highest nitrogen content and appeared to be the most soluble. It is also apparent that cold ethanol pretreatments were more effective than cold n-butanol treat- 5^ ments in facilitating lipid removal. Furthermore, the data reveal that cold euhanol was as efficient as room temperature n-butanol in achieving the removal of lipids from the membrane material (Table 3). and DellaMonica According to Morton (1950) and Zittle (1952)* n-butanol had specific ability to induce the separation of lipoprotein complexes. Zittle et a i . (1956b) used n-butanol to dissociate the fat-globule membrane material and the protein preparation contained 11.8 percent nitrogen. This value is about cne-half percent lower than reported values and Brunner (Weise and Palmer, 1934; Hare e1 al ., 1953a). et_ 1 954; The low nitrogen values obtained from n-butanol-treated membrane-proteins can be assumed to be due to an incomplete removal of lipids. Furthermore, the practice of using room temperature n-butanol on protein materials is open to question. Cohn (1943) and Taylor Leading authorities, such as (1955)* emphasize the importance of low temperatures whenever organic solvents are in contact with proteins. On the basis of the above discussion, ethanol was selected as a pretreatment technique prior to ethyl ether extractions for the removal of lipid from the fat-globule me m b r a n e. In the realm of blood chemistry, cold ethanol has fre­ quently been employed to disrupt lipoprotein complexes and Gardiner, 1910; McFariane, 1942; Cohn, Strong, Hughes, Mulford, Melin, and Taylor, 1946; and Jergensons, In this study, (Hardy 1955). lipid materials were not removed by McFar- l a n e ’s (1942) freezing-thawing technique. Apparently the lipid of the fat-membrane is more strongly complexed than 55 the lipids of human serum. During the preliminary investigations, the membrane proteins were decolorized frequently ducing the ether ex­ tractions. This presumably was due to the presence of per­ oxide groups in the ether. Moreover, when the ethanol was increased above 35 percent, the intensity of the brown color diminished and sometimes disappeared. The loss of the brown color was accompanied by a loss of xanthine oxidase activity. When 50 grams of concentrated lipoprotein were added to 100 milliliters of a 35 percent ethanol-ether solution, the final concentration was about 26 percent ethanol. Chemical, Physical and Biological Characteristics of the Fat-globule Membrane Proteins Electrophoretic characteristics. The electrophoretic mobility patterns presented in Figure II were selected from some of the exploratory runs. These patterns showed quali­ tative similarities when treated with 30 to 95 percent ethanol. Quite similar patterns were obtained for n-butanol-treated membrane except for a greater degree of diffusion. out the investigation, Through­ it was noted that the optical clarity of the protein solution was easier to achieve with the ethanol pretreated than with the n-butanol pretreated membrane pro­ teins. The skewed shape of these patterns indicated a heterogeneous protein system. An Inspection of the electro­ phoretic data in Table 8 reveals that the mobility of the major migrating peak was fastest in the Miller and Golder 56 (1950) buffer, which ranged from pH 2 to 12 and sodium chlor­ ide supplied 90 percent of the ionic strength. pared with this buffer system, In runs pre­ the false boundaries moved abnormally in the direction of migration. This is apparent in Figure 2 , row 4. Delta and epsilon boundaries were first thought to be due to slowly migrating protein or carbohydrate const!tuents. Longsworth and Maclnnes false boundaries. (1940) explained the presence of When an electrical current is passed through a TIselius cell, causing the descending boundary to move, the composition of the protein-containing buffer becomes changed In respect to the original buffer. Thus, an epsilon bound­ ary is formed between two solutions of the same buffer, each with a different concentration. The delta boundary on the ascending side Involved a gradient of protein concentration In addition to salt concentration gradient. Thus, the delta boundary is somewhat larger than the epsilon boundary. Buffers which contain large amounts of a neutral salt are frequently used In the electrophoresis of blood serum, whereas milk pro­ teins have been investigated generally in the more convention­ al types of buffers. Tap water-extracted membrane-protein appeared to be Identical with the protein extracted with buffer solution. This is Illustrated by the electrophoretic patterns in Figure II, row 1. When the unfractionated fat-globule membrane proteins were dispersed In a buffer solution and centrifuged before electrophoresis, a brownish-red insoluble pellicle was found in the bottom of the centrifuge tube. Thus, it was 57 suspected that the proteins in solution were not representa­ tive of the fat-membrane proteins. In the veronal buffer at pH 8 .8 , the soluble membrane proteins pretreated with 35 percent ethanol were most mobile. The proteins prepared with room temperature n-butanol had the lowest mobility. Since a decrease in mobility is indicative of changes In the protein, the assumption can be made that room temperature n-butanol produced undesirable changes in the protein system. t The electrophoretic mobility patterns in Figure III represent membrane proteins soluble in 0.02 M. sodium chloride solution. Various degrees of resolution were observed on either side of the Isoelectric zone. On the acid side, two and three components were resolved; whereas, on the alkaline side only one major skewed peak predominated. Apparently the soluble protein polymerized into various sized aggregates dependent on the pH of the buffer system. Thus, the apparent­ ly heterogeneous system on the acid side might have polymer­ ized into a more homogeneous unit on the alkaline side. At pH 5.30, a considerable amount of protein precipitated during dialysis and low mobilities were observed for the com­ ponents in the supernatant, indicating that the isoelectric area for the soluble membrane proteins was about pH 5*30. The fact that the mobilities were almost identical for the leading component of the diagrams in Figure II, row 4 and Figure III* row 8 indicated that lyophilization did not affect the soluble membrane proteins. The electrophoretic mobility patterns in Figure III approximate the patterns presented by Brunner et al^ (1953c). 58 The electrophoretic mobility patterns shown in Figure IV represent insoluble membrane-proteins treated with sodium sulfide. More protein precipitated from the buffer solution as the pH was lowered. Attempts to use acidic buffers resulted In the total precipitation of protein. Similar precipitations were encountered with proteins solubilized with detergents. When a solubilizing concentration of sodium sulfide was added to the proteins suspended In saline solution, the pH was 11.5. Accordingly, all electrophoretic work for the insoluble mem­ brane proteins was carried out In alkaline buffers. The mobil­ ity patterns indicate that sodium sulfide reduced the Insoluble proteins to a homogeneous mixture. This is Illustrated further in Figure IV, row 4, representing electrophoretic patterns of insoluble proteins solubilized with sodium lauryl sulfate and then treated with sodium sulfide. The effect of sodium lauryl sulfate and dodecyl benzene sodium sulfonate on the Insoluble membrane-proteins Is shown in Figure V With the exception of patterns in row 2, each run revealed two main components and a third, minor component. slower migrating, The minor component was removed by heating momentarily to 90° C . and is illustrated by the electrophoretic patterns in row 2. The heat-labile minor component might have been xanthine oxidase since this enzyme Is readily inactivated at 75°C . for five minutes (Zittle el a l ., 1955a). In row 3, sodium lauryl sulfate-solubilized material was examined in the Miller and Golder (1950) veronal-sodium chloride buffer. The results were quite similar to the patterns in rows 1 and 2 , particularly on the descending side. interestingly, the 59 false boundaries did not move abnormally as in Figure II, row 1, which indicates that the nature of the proteins affects the movement of the false boundary anomalies. The question arises as to whether the areas under the peaks are due to proteins or to detergent materials. However, experimental work revealed that sodium lauryl sulfate precipi­ tated when cooled to 0°c . Thus, the possibility was excluded that any of the peaks were due to sodium lauryl sulfate. In a veronal buffer of pH 8.6 and 0.10 ionic strength, Foster (19^9) reported a mobility of 18-19 * 10“^ cm.2 volt-1 sec.-1 for dodecyl benzene sodium sulfonate. This is almost double the mobility values reported for the solubilized proteins in Figure V, row K . Again, the possibility was excluded that a peak area represented dodecyl benzene sodium sulfonate. The number two peak (ascending side) in the sample treated with do­ decyl benzene sodium sulfonate seemed inclined to split Into two components. The possibility of a detergent-protein complex was in­ dicated In this study. First, an optimum detergent-protein ratio was needed to effect solubility. Second, the proteins did not precipitate in an alkaline medium during dialysis. The effect of sodium sulfide on the soluble proteins is shown in Figure VI, row 1. The patterns were diffused which indi­ cated that the proteins were broken into small units. Row 2 represents patterns obtained from a mixture of equal quan­ tities of the dlalyzed soluble proteins and sodium sulfidesolubilized Insoluble proteins. Interestingly, the electro­ phoretic patterns were unlike those for either the soluble proteins or solubilized insoluble proteins. However, the 60 mobility of the complex was similar to the leading component of the soluble proteins in a similar buffer. and Andrews (1951) Stanley, Whitnah suggested the possibility of interactions between various milk components. similar to those of row 2. Again, The diagrams in row 3 were the absence of the char­ acteristic spike for sodium sulfide-treated insoluble proteins was evident. Isoelectric zone for soluble proteins. Since it was not possible to Identify the same component in all of the electro­ phoretic patterns, the mobility of each component was selected for ordinate values in a pH-mobility plot. were considered in these calculations. Two separate trials Seven electrophoretic runs were made in the first trial and six in the second . Using the method of curvilinear regression, these points did not differ significantly from a straight line at the proba­ bility level of five percent, combined. therefore the 13 runs were The combined runs were also linear at the five percent probability level. The regression equation for the combined data is Y = 8.840 - 1.762X where Y represents mobil­ ity and X denotes pH. 0. Thus, pH 5.02. X Is equal to 5*02 when Y is equal to zero mobility for the leading component was at The original data for the leading component and its regression curve are shown In Figure X. The same statistical method was used to derive a regression equation for components two and three. For component two, the regression equation was Y = 7.250 - 1.455X. Thus, the point of zero mobility for component two was at pH 4.98. The third component had a regression equation of Y — 4.663 - 0.900X. The isoelectric 61 point for component three was then fixed at pH 5 .1 8 . and Weise electric (1933) and Jack and Dahle Palmer (1937a) reported the iso­ zone of membrane proteins to be at about pH 3.8 arid 4.3, respectively. These workers reported values for the un­ fractionated protein; therefore, their data should not be com­ pared with the above results. Solubility studies. The insolubility of at least a por­ tion of the fat-membrane proteins has been suggested by several workers (Hattori, 1925; Titus, Sommer and Hart, 1928; and Palmer and Weise, (1928), 1933). According to Titus, Sommer and Hart the membrane proteins were quite similar to casein. They believed that their preparation was contaminated with some unknown substance since the isolated protein would not dissolve In 0.5 N sodium hydroxide. Current knowledge suggests that these researchers isolated casein contaminated with mem­ brane proteins. Pedersen (193a) reported an insoluble mater­ ial in separator slime which was assumed to be related to casein. A more plausible explanation would seem to Indicate that the insoluble material was from the fat-globule membrane. The data in Table 4 illustrate the solubility character­ istics of the membrane proteins. The soluble fraction was readily soluble in aqueous solvents and was readily saltedout by half-saturation with ammonium sulfate. According to the classical protein classification systems, this indicates that the soluble fraction was ’’globulin” in nature. Sodium sulfide and thioglycolic acid readily solubilized the insol­ uble proteins. Since thioglycolic acid emitted noxious odors, this compound was not used further in the solubility studies. 62 The fact that these compounds were effective sis solubilizing agents suggests that the insoluble proteins have a disulfide linkage. Detergent-type chemicals have been found useful as deemulsifying agents in the preparation of butterfat 1962; and Stine and Patton, 1952). Patton (Patton, (1952) suggested that the lowering of interfacial tension probably facilitated the release of milk fat from the globules of milk. While this is probably a safe assumption, the possibility cannot be overlooked of a soluble complex formation between the deemulsifying agent and the insoluble membrane proteins. Thus, as the solubilized membrane proteins go into solution, the fat is freed. Stine and Patton (1952) reported that "of twenty-six agents which de-emulsifled cream quantitatively, twenty-four were of the cationic type, the other two being nonionic." and Patton However, King (1955 )> in citing the work of Stine (1952), referred to twenty-four anionics and two nonionics in discussing de-emulsifying agents. The surface- active agents used in this study were anionic with the ex­ ception of Triton X-100 which was nonionic. to note that Foster It is of interest (19^9) used dodecyl benzene sodium sulfon­ ate to solubilize zein prior to electrophoretic and sedi­ mentation velocity studies. Whenever sodium lauryl sulfate or dodecyl benzene sodium sulfonate was added to turbid aqueous protein suspensions, optical clarity was achieved in a few minutes. insoluble proteins heated in the presence of Triton X-100 remained tur­ bid until cooled to 6 2 ° C . Turbidity was again readily Induced by heating the mixture. This phenomenon indicated that the Triton X-100 protein complex had a negative critical solution temperature. Furthermore, proteins solubilized with Triton X-100 and Tergitol 7 were viscous and thus were rejected for electrophoretic studies. In addition, Tergitol 7 solutions became turbid upon cooling. Lyophilized insoluble membrane proteins did not yield to solubilization treatments. This held in the case of detergent-like chemicals and sodium sulfid The insoluble membrane proteins formed a stable suspen­ sion whenever these proteins were dispersed in aqueous solvent however, the proteins were readily precipitated upon freezing and thawing. Apparently, the Insoluble membrane proteins are sensitive to freezing temperatures. Enzyme studies. Enzyme activity was employed in this study as an indicator of undesirable changes in the protein system. Although this purpose was served by this criterion, other interesting observations were made. Table 5 reveals that phosphatase was concentrated in the soluble fraction whereas xanthine oxidase was concentrated in the insoluble fraction. Zlttle £t a l . (1956b) reported that freeze-drying caused a 15 and 46 percent loss of alkaline phosphatase and xanthine oxidase, respectively. Their data showed 40 units per milligram of phosphatase activity and 9 units per milli­ gram of xanthine oxidase activity on a dry-protein basis. When corrected for loss in enzymatic activity due to freezedrying, the samples used In this study were found to contain 49 and 145 units of phosphatase activity per milligram in the soluble portion, and l6 and 23 units of xanthine oxidase activity per milligram in the insoluble fraction. The fact that the phosphatase activity followed the soluble portion and xanthine oxidase activity was concentrated in the insol­ uble protein was of interest. This behavior indicated that the cold ethanol-ether treatments were specific in releasing phosphatase from the lipoprotein complex. This indicates that n-butanol is not unique in its ability to disrupt lipo­ protein complexes. Morton (1953, 195*0 concluded that the enzymatic activity of the fat-globule membrane was associated with microsomes adsorbed on the protein layer surrounding the fat globules, but this conclusion is not entirely supported by the data of other workers. Jenness and Palmer (19*f5a) found that the phospholipide/protein ratio in butter serum was higher than in washed cream buttermilk. This indicated that phospholipide were concentrated at the fat membrane interface. Furthermore, Zittle e_t a l . (l95ob) measured the xanthine oxidase and alka­ line phosphatase activity in a series of washed creams. The ratio of xanthine oxidase to alkaline phosphatase was 1 :1 .8 , 1 :2 .18 , 1 :2 .18 , and 1 :3-3 in four samples of washed cream, respectively. If xanthine oxidase and alkaline phos­ phatase were associated with a unit particle, one would expect the enzyme ratios in washed cream to remain constant during the wrashing procedure. Ball (1939) showed that the xanthine oxidase content of skimmilk increased as the milk aged, es­ pecially at low temperatures. Polonovski, Baudu and Neuzil (19^ 9 ) pointed out that freezing or the action of surfaceactive agents released xanthine oxidase from the membrane protein into true solution. In addition, these authors showed that a portion of the enzyme resisted the means of division employed and remained Intact on the membrane. From this discussion It seems clear that a more complete study is needed In this area of research. Chemical composition. The chemical composition of the fractionated membrane proteins was different than values reported for the entire protein. The values in Table 6 can be approximated to whole protein when the values 44 and 56 percent are used for the soluble and insoluble portions, respectively. On this basis, the whole protein contained 0.92 percent sulfur. percent sulfur. however, Weise and Palmer (193*0 reported 0 .96 Hare e_t a l . (1952) found 1.68 percent sulfur; this was not consistent with their values reported for the sulfur-containing amino acids. Their amino acid data accounted for only 0.64 percent sulfur. In this study, the amount of sulfur in the soluble portion calculated from the methionine and cystine data coincide exactly with the chemi­ cally determined sulfur. Methionine and cystine in the in­ soluble fraction accounted for 1.08 percent sulfur as compared to 1.03 percent sulfur determined chemically. That this fraction probably contained cysteine was indicated by its positive sulfhydryl reaction. The protein nitrogen values for the unfractionated mem­ brane ranged from 12.2 to 12.6 percent. The former value is more consistent with values reported previously (Weise and Palmer, 193'+; Hare et ad^, 1952; and Brunner et al^, 1953a). Since a portion of the proteins are water soluble, it is not 66 surprising that variable nitrogen values have been found. Whenever the membrane proteins were fractionated, the soluble portion always had a lower nitrogen content than the insoluble portion. Although this trend was invariant, the nitrogen values obtained varied among preparations for the two fractions. For the soluble fraction, nitrogen values ranged from 9.5 to 11.5 percent and from 12.9 to 13-9 percent for the Insoluble fraction. 6. A representative experiment is reported In Table The insoluble fraction approached the nitrogen content of a simple protein. Insoluble proteins prepared from lyophilized whole protein had a higher nitrogen value than that prepared from non-lyophilized protein. During the experiments, some of the in­ soluble proteins were freeze-dried and re-extracted with 0.02 M sodium chloride solution. After centrifugation the super­ natant was a yellowish-green fluorescent solution which was biuret-negative. Moreover, the nitrogen content of the in­ soluble proteins was increased. A value of 0.33 percent phosphorus was obtained for the unfractionated proteins by extrapolating from the soluble and Insoluble phosphorus contents. This value is in agreement with the data of Palmer and Weise (1933) who reported from 0.27 to 0.37 percent phosphorus. Separation of the membrane pro­ teins concentrated the phosphorus In the soluble portion - 0 .A6 percent as compared to 0.23 percent in the insoluble fraction. The high phosphorus value for the soluble fraction could not be correlated with nucleic acid phosphorus. 67 The data in Table 6 show that the insoluble fraction contained 7 -Oo percent ash as compared to 2.08 percent in the insoluble portion. This is eQuivalent to ^*3 percent ash for the whole membrane protein. This value is high when compared to 3-22 percent ash obtained by Hare et a i . (1952). Spectrographic evidence showed that the soluble fraction con­ tained considerably more phosphorus, magnesium, calcium, and copper than the insoluble proteins. The relatively low ni ­ trogen content of the soluble portion can be accounted for on the basis of a high mineral content and on the high sugar content as indicated by a strong,positive Molisch test. The intensity of the Molisch test varied among preparations of the insoluble fractions. Generally, a negative or very slightly positive Molisch reaction was noted. The Molisch reaction was especially weak or absent when the insoluble fraction was prepared from lyophilized proteins. The soluble protein solution reduced Fehling's solution after mild acid hydrolysis, but the quantity of cuprous oxide produced was small. Nevertheless, this indicated the presence of potential ketone or aldehyde reducing groups. Several attempts were made to chromatogram the sugars associated with the soluble membrane proteins. ridge For this purpose, the procedure of part­ (19^8) was adapted. technical difficulties, Most of the runs were fraught with the difficulties being intense heading and streaking on the chromatograms. sities, In spite of these adver­ presumptive evidence was obtained that pentose sugars and galactose or glucose were present. 68 Both protein fractions were nitroprusside-negative previous to heating. Following momentary heating of the In­ soluble fraction to 7 5 ° C ., a strong positive nitroprusside test was obtained. Apparently the Insoluble protein contained sulfhydryl groups which were activated by heat. was not observed with the soluble fraction. This reaction Insoluble pro­ teins treated for solubilization were sulfhydryl-negative. Presumably, the reducing ability of the sulfhydryl groups was lost due to some chemical or physical interaction of the detergent v/ith the proteins. Ultraviolet analyses. Characteristic absorption peaks in both fractions were most Intense at 278 millimicrons. Ab­ sorption in this region is characteristic for proteins con­ taining aromatic groups. Morton (195*0 reported the fat- globule membrane contained about 11 percent nucleic acids. In his method, acid-soluble phosphates were removed from the lipid free material by extracting with 0.5 N perchloric acid at 0 ° C . for four hours. The phosphorus which remained was assumed to be associated with nucleic acids and was extracted with 1 N perchloric acid at 8 0 ° G . Finally, he assumed that all absorption at 262 millimicrons was due to ribonucleic acid. 2 A Zi ttie et al . (1956b) were unable to find more than percent nucleic acid. Neither Morton (195*0 nor Zittle et a l . (1956b) have isolated and demonstrated the presence of purine or pyrimidine groups associated with the nucleic acids. Thus, the presence of ribonucleic acid in the membrane appears to be questionable. If nucleic acids were present In appre­ ciable quantities, Intense absorption peaks would be found 69 in the 260 millimicron region. Spectrograms in Figures III and IV do not show characteristic absorption In this region. The soluble and insoluble fractions from this study were ex­ amined by Zittle (1956) who was unable to demonstrate the presence of nucleic acids. The fact that nucleic acids might not be associated with the membrane seriously poses M o r t o n ’s (1953, 195*0 microsome theory. According to Novikoff, and Haurowltz nucleic acids. Podber, Ryan and Noe (1953) (1955), microsomes definitely contain ribo­ More data are needed before concluding that the fat-globule membrane proteins contain nucleic acids. Evidence obtained in this study appears to preclude the presence of nucleic acids as a part of the membrane proteins. Infrared analyses. The principle reason for obtaining an infrared analyses was to determine whether any differences were manifested between the two fractions in the absorption spectra. The only difference found was that the soluble portion showed a slight shoulder at 5-75 microns. Absorptions at 3 .^5 , 6 .8 5 , and 7-27 microns were attributed to functional groups of the mineral oil in the Nujol mull. The peaks at 3.05 and 6.05 microns were due to amino stretch and bend, respectively. The absorption area at 9*59 microns was pro­ visionally assigned to aryl phosphate linkages. Bellamy's (195*0 text was used to identify the absorption areas. AminO acids. Adjustment of the amino acid values to a whole membrane protein basis and subsequent comparisons with literature data authenticated the experimental values. is illustrated in Table 7* This Brunner et a l . (1953a) and Hare 70 a l - (1952)reported 1.5 percent cystine for the membrane proteins, whereas a value of 2.^3 percent was found In this study. This difference mignt be explained on the basis of different hydrolysis procedures. The method used in this study was a special procedure for cystine, adapted from Horn and Blum (1956), in which the proteins were hydrolyzed in an autoclave at 1 1 5 ° G . for only 30 minutes with 20 percent hydrochloric acid . Both the soluble and insoluble fractions had amino acid compositions quite different from other reported milk pro­ teins . The low glutamic acid and high arginine content of the insoluble proteins is unlike that of any other milk pro­ tein. Block and Vickery (1931) reported that proteins can be classed as keratins which are insoluble in dilute acids, alkalies, water, and organic solvents, but when hydrolyzed with acid yield a*l : k :12 ratio of histidine, arginine. Block and Bolling lysine, and (1939) classified Insoluble protein which possessed a 1:1 ratio of lysine to arginine as pseudokeratins. For the insoluble protein preparation, a 1:1 ratio of lysine to arginine was found (Table 7)- Thus, on the basis of insolubility, reaction to specific chemical reagents and amino acid composition, the insoluble fraction can be provisionally classified as a pseudokeratin. Certainly one can postulate that a certain amount of protein, resistant to inherent proteolytic enzymes and insoluble in the milk serum, might surround and stabilize the fat globules. The amino acid composition of the soluble fraction was unlike any of the known milk proteins. This fraction contained 71 more cystine and less glutamic acid than any of the reported milk proteins. Interestingly, the soluble fraction contained quantitatively less amino acids than the insoluble fraction. However, the soluble fraction contained considerable non­ protein materials which might have contributed to the destruc­ tion of amino acids upon hydrolysis. Actually, the amino acid composition of the two fractions was not radically dif­ ferent. The greatest difference between the fractions was found in the arginine, methionine, and valine contents. Quantitative determination of amino acids by paper chromatography is relatively new, but the accuracy of this method has been substantiated by Block and Weiss (1955). The importance of using good analytical reagents and satur­ ating the chromatographic chamber before the run was made in this study. The values obtained for phenylalanine and proline were questionable and are not reported, but the method appears to be adequate for these determinations. With the exception of proline, the color of the developed chromato­ grams was quite stable when stored in the dark. Proline produced a green, unstable color when developed with isatin which could not be eluted with the extractant used. Appar­ ently a densitometer should be used in conjunction with pro­ line determinations. Although the amino acid composition of the soluble frac­ tion did not permit classification of this protein, there are data which Indicate that it Is globulin in nature. the basis of sedimentation velocity studies On (S^o = 7*3) > 72 Brunner et_ a l . (1953b) provisionally classified the fat-mem­ brane protein as globulin-like. Some exploratory studies (unreported) of the soluble proteins with a Spinco Model E Ultracentrifuge substantiated the above data; however, a low molecular weight component of low concentration was also ob­ served. Thus, on the basis of sedimentation studies, Insol­ ubility in half-saturated ammonium sulfate, and an isoelectric zone near pH 5.0, the term globulin-like best fitted the soluble fraction. The primary purpose of determining amino acid composition of the two fractions was to compare both fractions and the Individual fraction compositions with other known proteins. Since the nitrogen values of each fraction indicated the presence of non-protein materials, minimum molecular weights were not calculated. Spectrographlc analyses. The residue from "Che ash deter­ minations was taken-up in 1:1 hydrochloric acid and submitted for spectrographlc analysis in the carbon arc of a Hilger spectrograph. A Jarrell-Ash microphotometer was used to evaluate the spectrograms in which cobalt was the internal standard. Qualitative and approximate quantitative data for identified minerals are given in Table 9* All of the elements reported here have been demonstrated spectographically in whole milk (Dingle and Sheldon, 1933). also reported rubidium, milk. These researchers lithium, barium and strontium In whole Under the conditions of this study, these four elements could not be demonstrated in the membrane proteins. Gehrke, 73 Baker, Affsprung and Pickett (195*0 reported seml-quantltative spectrographlc data for the trace elements In whole milk. In the instances where comparisons were possible, the fatglobule membrane proteins possessed higher concentrations of specific elements than those reported for whole milk. This indicates that many of the trace elements in whole milk are concentrated in the membrane. Calcium was found in the fat-membrane material by Hattori (1925), this finding. but Palmer and Weise (1933) could not confirm These workers believed that all of the calcium present was dialyzable. Calcium was detected spectrograph- ically in this study in proteins which had been exhaustively dialyzed against distilled water. were a contaminant, one in each fraction. Moreover, if the calcium would expect to find equal quantities As with most elements associated with proteins, the role of calcium in the membrane proteins is obscure. On the basis of known biological activity associated with the membrane, the identification of iron, magnesium and molybdenum was not surprising. Richert and Westerfeld (1953) isolated xanthine oxidase from cream which contained 0 . 0 3 percent molybdenum. It is interesting to note that xanthine oxidase and molybdenum were both concentrated in the insoluble proteins. Richert and Westerfeld (1 953) believed that moly­ bdenum was a part of the xanthine oxidase molecule. Morton (195*0 reported that cytochrome-c was associated with the membrane proteins; thus, the presence of iron was suspected. Probably the concentration of magnesium in the soluble fraction 74 was related to Its high phosphatase activity. A limited amount of Information is in the literature showing a complete elemental composition of milk protein residue ash. The data in Table 9 represent the first spectro- graphic analysis of the membrane proteins and indicate a wide difference in the mineral distribution between the two fractions. Although the value of these data is of limited use, their potential significance can not be predicted. SUMMARY AND CONCLUSIONS Essentially, this research was divided into two sections: first, to isolate the fat-globule membrane proteins with a minimum of undesirable changes, and second, to study the physical, chemical, and biological characteristics of the isolated proteins. Five concentrations of cold ethanol, four of cold n-butanol, and room temperature n-butanol were investigated as agents to disrupt lipoprotein complexes. Proteins pretreated with 35 percent ethanol (final concen­ tration about 26 percent) were most amenable to physico­ chemical studies. Under the conditions of this study, ethanol pretreatment was more satisfactory than either cold or room temperature n-butanol for disrupting the membrane lipoprotein complex. The membrane proteins were separated into soluble and insoluble fractions based upon solubility in a 0.02 M sodium chloride solution. These fractions differed widely on the basis of physical, chemical and biological properties. The soluble fraction had a strong Molisch reaction, reduced Fehling's solution subsequent to mild acid hydrolysis, and was sulfhydryl-negative after heating to 75°C. Nitrogen values varied with individual preparations from 9*5 to 11.5 percent. An average of seven-fold more phosphatase activity was found in the soluble than in the insoluble fraction. A single skewed component was observed in alkaline buffers, whereas three closely associated components were evident in 75 76 acidic buffers when the soluble fraction was studied electrophoretically. Regression equations Y » 8.840 - I.762X, 7 — 7*250 - 1.455X, and Y = 4.663 - 0.900X were calculated which gave isoelectric points at pH 5.02, 4.98, and 5.18, respectively. On the basis of sedimentation velocity data, insolubil­ ity in half-saturated ammonium sulfate and an isoelectric zone near pH 5-0, the soluble proteins were tentatively classified as globulin in nature. The residual bases, 25 percent fraction was insoluble in dilute acids and sulfuric acid and 6 and 8 M urea. Strong reducing agents commonly used to attack disulfide-linked proteins and certain detergents were found as capable solu­ bilizing agents. Sodium sulfide and sodium lauryl sulfate were found to be good solubilizing agents. The nitrogen for values ranged from 12.9 to 13*9 percent the insoluble protein. A qualitative sulfhydryl-posltlve reaction was obtained upon heating the protein moiety to 75°G. In contrast to the white color of the soluble fraction, the insoluble material was a reddish-brown. The insoluble fraction contained 5-6 times more iron, 25 times more molyb­ denum, and 10 times more xanthine oxidase activity than the soluble fraction. Electrophoretic analyses of the insoluble fraction were carried out on solubilized-proteln in alkaline buffers. Sodium sulfide-solubilized material showed one homogeneous component and the detergent-solubilized protein had two 77 prominent and one minor components. On the basis of insolubility in the usual protein solvents, reactivity to specific reducing agents, and amino acid composition, the insoluble proteins were provisionally classified as pseudokeratin in nature. LITERATURE CITED Abderhalden E., and Voltz, W. 1909. Beitrag zur Kenntnis der Zusammensetzung and der Natur der Hullen der Milchkugelchen. HoppeS e y l e r ’s Ztschr. physiol. Chem., 59:13-18. Ascherson, 1840. ’. M. On the physiological utility of the fats and on a new theory of cell formation based on their cooperation and supported by several new facts. A translation by Emil Hatschek in The Foundations of Colloid Chemistry, pp.13-27. L o n d o n ; E. Benn.~ Ltd .“ (1955 .“P Avis, P. G. Berg e l , F., and Bray, R. C. 1955. Cellular constituents. The chemistry of xanthine oxidase. Part I. The preparation of a crystalline xanthine oxidase from c o w ’s milk. J. Chem. S o c ., pp. 1100-1105. Babcock:, S. M. Fibrin In milk. 1889. R p t ., 63-6 8 . Wis. Agr. Expt. S t a ., 6 th Ann. B a l 1, E ■ G • Xanthine oxidase: Purification and properties. 1939. J. Biol. Chem., 128:51-87. Bellamy, L. J. The Infrared spectra of complex molecules. John 1954. Wiley and 3ohs8 Inc ., New Y o r k T N T T T 322 pp. Block:, R. J ., and Bolling, D. The amino acid composition of keratins: The 1939. composition of gorgonin, spongin, turtle scutes, and other keratins. J. Biol. Chem., 127:685-893. Block, R. J ., and Vickery, H. B. The basic amino acids of proteins. A chemical 1931. relationship between various keratins. J. Biol. Chem., 93:113-117* Block, R. J ., and Weiss, K. W . Studies of bovine whey proteins. IV. The amino 1955. acid analyses of crystalline p -lactoglobulin and -<-lactalbumin by quantitative paper chromatography. Arch. Blochem., 55:315-320. 78 79 Brunner, J . R., Duncan, C. W., and Trout, G. M . 1953a. The fat-globule membrane of nonhomogenized and homogenized milk:. I. The isolation and amino acid composition of the fat—membrane proteins. Food Research, 18:454-462. Brunner, J . R., Duncan, C. W., Trout, G. M., and MacKenzie, M. 1953b. The fat-globule membrane of nonhomogenized and homogenized milk. III. Differences in the sedimentation diagrams of the fat-membrane proteins. Food Research, 18:1-6. Brunner, J . R., Lillevik, H. A., Trout, G. M., and Duncan, C .W. 1953c. The fat-globule membrane of nonhomogenized and homogenized milk. II. Differences in the electro­ phoretic patterns of the fat-membrane proteins. Food Research, 18:463-468. Bull, H. B 1951. Cohn, E. J 1943. Physical Biochemistry. John Wiley and Sons, Inc., New York,”~N. Y 355 PP* The solubility of proteins. Proteins, Amino Acids and Peptides as Ions and Dipolar Ions. Edited by Cohn, E . T7, and ^Tdsall, J 7 T. Rheinhold Publishing Corporation, New York, N . Y . 686 p p . Cohn, E. 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