Fractionation of Skimmilk Casein Micelles by Rate-Zone and Isopycnic-Zone Ultracentrifugation in Sucrose Gradients1

Fractionation of Skimmilk Casein Micelles by Rate-Zone and Isopycnic-Zone Ultracentrifugation in Sucrose Gradients C. V'. MORR, S. If. C. LIN, and R. V. JOSEPH$ON 2

Department of Food Science and Industries, University of Minnesota, St. Paul 55101 Abstract

Rate-zone and isopycnic-zone ultracentrifugation techniques with sucrose gradients in a swinging-bucket rotor were utilized for fractionating skimmilk proteins with emphasis upon characterizing the different size casein micelles. Rate-zone ultracentrifugation separated skimmilk casein micelles from soluble casein and whey proteins, and further fractionated the different size micelles. Up to , - 9 2 % of skimmilk casein mieelles was sedimented to the bottom of a 17.2 to 27.5% w / v sucrose gradient by centrifuging at 206,000 × g for two hours at 0 to 5 C. Zonal electrophoresis data, uncorrected for differences in dyebinding coefficients, revealed that the smallest casein micelles and soluble casein contained ~ 8 . 8 % as-casein, 89% fl-casein and 2.2% ~-easein and the largest micelles recovered in the pellet fraction contained ~ 8 0 % as-casein , 19% fl-casein, and 0.8% ~-casein. Isopycnic-zone ultracentrifugatiou handed whey proteins and soluble casein in ~ 8 to 10% w / v sucrose (p = 1.02 to 1.04 g / m l ) , but failed to band casein micelles even in =~70% w / v sucrose gradients. I t was concluded that the high concentrations of sucrose increased buoyant density of the casein mieelles by removing their solvation and hydration layers, and thus, the isopyenie-zone technique is not applicable for studying casein mieelles in their native state. Introduction

the large and small micelles are initially distributed throughout the entire medium, and in addition, convectional disturbances produce mixing of the different size fractions during sedimentation (1). Repeated resuspension and sedimentation of the pellet fraction to improve the resolution of different size micelles is not satisfactory because the tightly-packed pellet is not completely redispersed in the absence of protein dissociating agents, and also, the composition of the micelles is altered by removing exposed inorganic and casein components (15). Sucrose gradient ultracentrifugation involves fractionating protein particles in a sucrose gradient according to differences in size (ratezone) or buoyant density (isopycnic) properties (2). The rate-zone technique offers special promise for resolving different size micelle fractions because they sediment from a thin sampie zone at characteristic rates through a medium stabilized against convectional mixing. Tessier et al. (17) used a sucrose density gradient technique in a zonal rotor to fractionate skimmilk proteins by differences in their banding densities. Our p a p e r describes, in greater detail than before (13), experiments to adapt the rate-zone ultracentrifugation technique in a swinging-bucket rotor to fractionate skimmilk proteins, especially the different size casein micelles. Limited isopycnic-zone ultracentrifugation experiments to establish the buoyant density characteristics of micellar and nomnicellar soluble skimmitk proteins are also described. Materials and Methods

Sample preparation. Skilmnilk was separated from University herd milk by warming to 35 to 40 C and centrifuging at ,~1,000 × g for 15 to 20 rain at least 24 hr before use. Colloidal phosphate-free ( C P F ) skimmilk was prepared by adding 1.4 mmoles disodium E D T A and 2.1 mmoles tetrasodium E D T A per 100 ml Received for publication November 27, ]970. skimmilk and dialyzing against several changes Scientific Journal Series Paper 7485, Minnesota of an excess of milk for 48 to 72 hr at 0 to 5 C (7). Acid whey was prepared by adjusting p H Agricultural Experiment Station. 2 Present address: Department of Dairy Tech- of the skimmilk to 4.6 with z¢ HCI at 35 to nology, The Ohio State University, Columbus 40 C and centrifuging at -~1,000 X g or higher to sediment the casein precipitate. 43210. 994

As documented in a recent review by Decelles (3), differential ultracentrifugation has been widely used to prepare different size casein micelle fractions. However, this technique does not provide precise size fractionation because

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Sucrose gradient preparation. Gradients were prepared in sinmlated milk ultrafiltrate (SMUF) to minimize changes in casein micelles during fraetionation. Since addition of sucrose to SMUF solution lowered its p H and salt concentration, the dry-blended salt mixture (6) and sucrose were dissolved in water and the p H adjusted to 6.6 just before making to final volume at 20 C. Linear gradients were formed in 12 mm id × 96 mm centrifuge tubes at a final volume of 8 ml, with a model 570 ISCO gradient former s or an Auto-Gradient device. 4 The linearity and limiting sucrose concentrations of the gradients were verified in separate experiments by incorporating Anfido Black dye into the heavy sucrose solution and monitoring the gradient at 640 nm by the speetrophotometrie procedure used to examine protein fractions. Sucrose gradients are reported per cent by volume and densities correspond to 5 C sucrose in water solutions taken from Handbook of Biochemistry (5). Sample layering. Skimmilk protein samples were carefully layered onto the gradients with either a hypodermic syringe or a fine-bore pipette° Samples of 0.02 ml were used for 280 nm monitor experiments and larger volumes of 0.2 to 0.5 ml were used to obtain sufficient protein for quantitation and zonal electrophoresis.. Although this layering technique provided satisfactory results for whey proteins and soluble casein, casein micelles diffused into the upper region of the gradients upon standing ~'or 30 rain or longer. Centrif~gation. Gradients were centrifuged at 206,000 × g (maximum) in a B-35 preparative ultracentrifuge 5 with an SB-269 swingingbucket rotor at 0 to 5, 10 or 20 C. Average rotor speeds were read from an electronicallysynchronized tachometer. The rotor was accelerated at the maximmn rate and decelerated by eoasting without the brake. Equivalent times for the acceleration and deceleration periods~ determined by integrating the area under a graph of rotor speed-squared versus time wRh a planimeter, are included in all centrifugation times. Gradient re~wval. A rubber stopper, fitted with two different length 18 gauge syringe needles, was tightly inserted into the top of the centrifuge tube so that the tip of the longer needle was near the bottom of the tube and 3 lnstr amentation Specialties Co., Lincoln, Nebraskm 4 Chromatography Corporation of America, Carpentersville, Illinois. 5 International Equipment Company, Needham Heights, Massachusetts.

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the tip of the shorter needle was just above the meniscus. A heavy sucrose solution, whose concentration was preadjusted to 5 to 10% higher than that at the bottom of the gradient, was slowly pumped through the longer needle with a syringe pump 6 to lift the gradient and force it out through the shorter syringe needle and 1.2 m m i d tygon tubing' connected to the speetrophotometer and fraction collector. Examination of protein fractions. The gradients were monitored at 280 nm in a DB spectrophotometer, 7 collected in fractions, and examined for protein content by the Folin method of Lowry et al. (9). Corresponding fractions from up to 12 replicate gradients were combined and examined by zonal electrophoresis to determine the distribution of protein components. Fractions were adjusted to p H 4.6 at 35 to 40 C and centrifuged at ~1,000 × g to sediment the casein precipitate. Casein components in the above sediment fractions were washed once with water, freezedried, and examined by vertical starch gel electrophoresis s by described procedures (12). Gel patterns were scanned by a Photovolt model 542 Densicc~rd9 with a 620 mn filter and the peak areas of the patterns were determined with an attached ~.[odel 49 automatic integrator2 Experimental Results

Rate-zone z~ltracentrifrgation. Tile important conditions affecting fractionation of skimmilk proteins, especially casein micelles, by ratezone ultracentrifugation were investigated. Whey proteins and nomnicellar casein remained in the top 0.5 ml fraction of the gradient (Peak 1 in Fig. 1) when whey, CPF skimmilk and skimmilk were centrifuged 206,000 X g for 25 rain on 17.2 to 27.5% sucrose gradients. Small and intermediate size casein micelles in skimmilk were distributed throughout the gradient as a broad peak (Peak 2 in Fig. lc) and tile larger casein micelles sedimented through the gradient to form a pellet which was not shown in the monitor pattern. Peak :~ was produced by a combination of the schlieren effect at the boundary between the gradient and the heavy sucrose solution used to remove the gradient and also large casein micelles concentrated in the bottom gradient fraction. The effect of varying centrifugation time and sucrose concentration in the gradients upon 6 Sage Instruments, White Plains, New York. 7 Beckman Instrmnents, Inc., Fullerton, California. s Buehler Instruments, Inc., l~ort Lee, N.J. 9 Photovolt Corporation, New York, N.Y. ~OUICNAL Olt~ DAIRY SCIENCE VOL. 5{, NO. 7

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Fla. 1. Sedimentation profile of skimmilk proteins: sample, 0.2 ml, 0 to 5 C; a) pit 4.6 whey; b) CPF skimmilk; and c) skimmilk; gradient, 17.2 to 27.5% sucrose; centrifugation, 206,000 × g for 15 min at 0 to 5 C. sedimentation profile is shown in Figure 2. Satisfactory fractionation of casein micelles from nonmicellar casein and whey proteins was achieved on 17.2 to 27.5% sucrose gradients centrifuged at 206,000 × g for 15 rain (Fig. 2b). Similar fractionation was obtained with 34.2 to 44.5% sucrose gradients centrifuged at 206,000 × g for longer times, i.e., 35 and 65 rain (Fig. 2d and e). Centrifugation of 17.2 to 27.5% sucrose gradients for more than 15 rain or 34.2 to 44.5% sucrose gradients

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FIG. 2. Sedimentation profile of skimmilk proteins: sample, 0.2 ml 0 to 5 C skimmilk; centrifugation, 206,000 X g at 0 to 5 C; gradient and centrifugation time, a) 8.6 to 13.7% sucrose for 15 min; b) 17.2 to 27.5% sucrose for 15 rain; e) 34.2 to 44.5% sucrose for 15 min; d) same as e for 35 min; e) same as e for 65 min; and f) 34.2 to 52.4°~ sucrose for 35 mira JO~JI~NAL O F D A I R Y S C I E N C ~ ~OL. 54, ~K~O. 7

longer than 65 rain did not produce noticeable sedimentation of whey proteins and soluble casein (Peak 1) from the meniscus zone of the gradient, but sedimented progressively greater amounts of casein micelles into the pellet (see below). These results are consistent with the observed banding density values for whey proteins and soluble casein of 5% sucrose reported by Tessier et al. (17). Use of 8.6 to 13.7% sucrose gradients was less satisfactory because their low densities of 1.03 to 1.05 g/ml did not adequately stabilize the sample layer at the meniscus, and thus, resulted in poor resolution of Peaks 1 and 2 (Fig. 2a). Trials with higher concentration gradients, i.e., 34.2 to 65.4% sucrose, demonstrated that their high densities of 1.132 to 1.247 g/ml retarded sedimentation of casein micelles (Fig. 2f), and therefore were unsatisfactory for rate-zone ultracentrifugation. Additional sedimentation profile data not shown indicated that ultracentrifugation at forces below 206,000 X g for correspondingly longer times provided comparable fractionation of micellar and nonmicellar skimmilk proteins. A histogram of Folin protein values for gradient fractions in Figure 3 agrees with the sedimentation profiles obtained under the same experimental conditions as in Figures lc, 2b, and 4a. The data also confirm that most of the whey proteins are recovered in the top 0.5 ml fraction of the centrifuged gradient. The histogram revealed less sharply defined peaks than the 280 nm monitor patterns because larger sample layers were required in the former technique and the latter technique possesses inherently greater sensitivity for detecting minor fluctuations in protein concentration within narrow regions of the gradient. Casein micelles in skimmilk equilibrated at 37 C were more completely sedimented than those equilibrated at 20 C when both were centrifuged on 20 C gradients (Fig. 4). These findings demonstrate the capability of the ratezone ultracentrifugation technique for studying the temperature-induced aggregation-disaggregation reaction of casein micelles. The less complete sedimentation of the 0 to 5 C skimmilk casein micelles on 0 to 5 C gradients than above is due to disaggregation of casein micelles as well as higher viscosity and density of the gradient at low temperature. From the aforementioned experiments, relatively shallow 17.2 to 27.5% sucrose gradients and centrifngation at 206,000 × g for short times, i.e., 15 rain, were selected as standard conditions for the rate-zone ultracentrifugation techniques. Densitometer data for starch gel electro-

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FIO. 3. Histogram of whey proteins ( . . . . ) and casein ( - - ) concentrations in sucrose gradient fractions:sample, 0.5 ml 0 to 5 C skimmilk; gradient, 17.2 27.5% sucrose; centrifugation, 206,000 × g for 15 min at 0 to 5 C. phoresis patterns of rate-zone ultracentrifugation fractions of skimmilk casein in Figure 5 substantiate those in Figure 1 that nonsedimenting, soluble casein is fractionated from micellar casein and that casein mieelles are further fractionated among the different gradient fractions and pellet. Soluble casein, in the top 0.5 ml gradient fraction, contained ~ 8 9 % fi-casein and, in this regard, resembles serum prepared by ultracentrifuging skimmilk at ~90,000 × g. The progressively larger casein mieelles in the fractions closer to the bottom of the gradient and pellet contain correspondingly more as-casein and less fi- and Kcasein. It should be noted that these densitometer data have not been corrected for differences in Amido Black binding by the casein species. Although the present casein distributions for

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I~IG. 4. Sedimentation profile of skimmilk proteins: gradient, 17.2 to 27.5% sucrose; eentrifugation, 206,000 × g for 15 rain; sample, 0.2 ml; temperature, a) 0 to 5 C skimmilk centrifuged at 0 to 5 C; b) skimmilk held at 20 C for one hour and centrifuged at 20 C; and c) skimmilk held at 37 C for one hour and centrifuged at 2O C.

standard casein, i.e., 54% as- , 41% fl-, and 5% K-casein, agree with data from numerous densitometer experiments in our laboratory and with those of Thompson (18), the composition of whole casein is probably ,-~50% as-, 33% fi-, and 15% K-casein (16). Thus, although the present casein distributions are admittedly low in K-casein and high in a~- and fi-caseins, comparison of these data with those of Rose et al. (:[6) confirms that the present rate-zone ultracentrlfugation technique provides considerably better resolution of the different size skimmilk casein micelle fractions than by preparative ultracentrifugation. The present data also confirm that the different size skimmilk casein micelles contain dissimilar distributions of the casein species, which has been a point of contention for some time (3). Data in Figure 6 reveal that ~ 8 0 % of total casein, i.e., 92% of the casein micelles, was sedimented into the pellet and bottom one milliliter fraction of 17.2 to 27.5% sucrose gradients by centrifuging at 206,000 X g for t w o hours at 0 to 5 C. However, only ~ 5 0 % of the total casein, presumably the larger and denser mieelles, was recovered in the pellet fraction. Those casein micelles that concentrate in the bottom gradient fraction are probably nmre highly solvated than those that pack into the pellet. These differences in degree of solvation may be due to the variations in casein composition noted already for the gradient and pellet fractions. Electron microscopic examination of centrifuged gradient fractions confirmed that the progressively larger casein micelles were sedimented correspondingly greater distances and thereby recovered in lower fractions of ~OURI~AL OF DAIRY SCIE2¢CE ~OL. 54, NO. 7

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60 to 70% sucrose gradients. Soluble casein and whey proteins formed a broad band (Peak 2) centered at ~ 8 to 10% sucrose (p = 1.03 to 1.04 g / m l ) , however, contrary to previously published data (17), casein micelles (Peak 3 plus pellet) did not band in --<70% sucrose zones (p = 1.267 g / m l ) but continued to sediment into the pellet fr~etion.

Discussion Rate-zone ultracentri£ugation allows for more convenient and precise fractionation of different size casein micelles than by preparative ultracentrifugation. Six gradients can be centrifuged simultaneously in a swinging-bucket rotor to enable examination of different samples or gradient conditions by the monitor technique, or to obtain sufficient amounts of corresponding casein micelle fractions from replicate gradients for analysis. Automated techniques for forming and handling the gradients provide speed, convenience, and reproducibility to the method, i.e., the entire procedure from forming the ga'adient through collecting the fractions can normally be completed within 60 to 90 min. This analytical technique, which fraetionates up to 10 to 15 mg casein micelles per gradient, provides a useful analytical complement to preparative sucrose gradient fractionation of casein micelles by zonal ultracentrifugation. F o r example, centrifugation and gradient variations, conveniently evaluated by the present technique, can be readily adapted to preparative casein micelle fractionation by zonal ultraeentrifugation. The sedimentation rate of spherical casein FIG. 5. Starch gel electrophoresis and densitometer data of casein components in sucrose gradient fractions: sample, 0.2 ml 0 to 5 C skimmilk; gradient, 17.2 to 27.5% sucrose; centrifugation, 206,000 × g for 15 mln at 0 to 5 C; successive 0.5 ml fractions (1 to 13) collected in order from the top of the gradient; P, pellet; CS, casein standard. Per cent casein distributions determined by densitometry, were uncorrected for difference in dye binding of the casein species. The 5'-casein zone probably contains a number of minor casein components (12). the gradient by rate-zone ultraeentrifugatiou (8). Isopycnic-zone ultracentrifugation. The isopycnie-zone ultraeentrifugation technique was evaluated for fractionating soluble and micellar skimmilk proteins and obtaining more precise data on their buoyant densities for calculating sedimentation coefficients (S2o.w) by the ratezone ultracentrifugation. Results in Figure 7 are for 16 hr centrifugation experiments with 0 to 5 C skimmilk on 0 to 20, 0 to 70, and JOURI~ALOF DAIRY SCIENCE¥OL. 54, NO. 7

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FI(~o 7. Isopycnie-zone ultraeentrifugation profile of skimmilk proteins: gradient, a) 0 to 20% sucrose; b) 0 to 70% sucrose; and e) 60 to 70% sucrose; centrifugation, 206,000 × g for 16 hours at 5 C; sample, 0.05 ml 0 to 5 C skimmilk. Shallow gradients a and e were used to provide better resolution of soluble casein and the heavier casein mieelles, respectively, than that in gradient b. Dotted lines connect corresponding regions of the separate gradients. micelles by centrifugation through a sucrose gradient can be expressed by Stokes' law: dx dt

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where; x is the distance the micelles sediment in time t; r, the radius of the nfieelles; p,, the density of the micelle; Pm~ the density of the gradient medium; g, the acceleration due to centrifugal force; and V, viscosity of the gradient medimn. Experimental conditions were selected to maximize differences between the density of the nficelles and the gradient medium and to minimize the increase in viscosity and density from the top to the bottom of the gradient. These objectives were met with mininml sucrose concentrations at the top of the gradient to stabilize casein-free skimmilk serum with a reported density of 1.0264 g/ml (4) and with density gradients which arc sufficiently steep to prevent mixing during' eentrifugation and subsequent handling, and yet shallow enough to not unduly impede the sedimentation of casein micelles with densities in the order of 1.114 (4) to ~1.212 (17) g/ml. Although

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the continuously increasing density and viscosity of the gradient medium gradually reduce the micelle sedimentation rate, the proportional increase in centrifugal force with increasing radius has a balancing effect, and therefore, the decrease in sedimentation rate with sedimentation distance through the gradient is less than expected (10). ~{ethods for correcting these factors in calculating sedimentation coefficients for casein micelles in sucrose gradients will be the subject of a later paper. The inability of tile isopycnic-zone ultracentrifugation technique to band casein mieelles at sucrose concentrations of ~ 7 0 % (p ~ 1.267 g/ml) in the present study is in contrast with the findings of Tessier et al. (17) who reported that casein micelles banded in 55% sucrose (p = 1.212 g/ml) after centrifuging for only three hours in a zonal rotor at 5 C. Although the reported density of casein micelles dispersed in their native skimmilk serum is reported by Ford et ah (4) to be ~1.114 g/ml, this value is based upon a number of important assumptions that may not apply to casein micelles dispersed in high sucrose concentration solutions. Ford et al. (4) assumed that the dry micellar casein complex had an average apparent specific volume of 0.697 ml/g, the density of the hydration water (0.52 g/g dry complex) was equal to that of free water, the solvate liquid was equivalent to casein-free serum with a density of 1.0264 g/ml at 25 C, and each gram of dry complex occupied 3.1 nd (volmninosity) when dispersed in skimmilk at 25 C. Whitnah and Rutz (20) obtained similar voluminosities of ~3.00 ml/g for casein micelles dispersed in skimmilk serum at 25 C and further showed that casein mieelle voluminosity is strongly influenced by temperature. Applying Ford's equation (4) to the voluminosity data of Whitnah and Rutz (20), and assuming that the degree of hydration remains 0.52 g/g dry complex and that the density of the hydration and solvation liquids remains constant leads to the following densities of casein mieelles in skimmilk serum. Tempera¥olumiture nosity Density

(c) (ml/g) (g/ml) -~ 6.45 1.068 20 4.06 1.082 25 3.00 1.116 37 3.36 1.106 The voluminosity of casein micelles is probably reduced at higher temperatures by lowering the degree of hydration and the amount of associated solvate liquid both surrounding and JOURNAL OF DA.I~Y SCIENCE ~OL. ~4, NO. 7

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within the porous interior of the micelles (11, 14). The aforementioned findings substantiate the importance of temperature and other medium conditions in determining the voluminosity, density, and solvation of dispersed casein micelles (19). Using F o r d ' s equation (4), it can be calculated that removing the solvation layer without altering hydration would result in a limiting theoretical casein micelle density of ,-1.247 g / m l . Replacement of the solvate liquid layer with a 70% sucrose solution (p 1.267 g / m l ) without altering voluminosity and hydration would result in a theoretical limiting micelle density of ~1.263 g / m l . Removal of both solvate and hydrate liquids would result in a theoretical limiting density for the dispersed micelles of ~1.434 g / m l . Although no direct evidence was obtained in this study, the failure to completely band casein micelles in --~70% sucrose gradients strongly suggests that high sucrose concentrations probably increase the buoyant density of casein micelles f r o m ~1.114 g / m l (4) to ~ 1 . 2 6 7 g / m l by reducing their degree of solvation and hydration or replacing the solvation layer with dense sucrose solution. Electron microscopic examination of casein micelles recovered f r o m high sucrose concentration gradients confirmed that they were altered in appearance, i.e., they appeared nonspherical and partially aggregated (8). Although casein micelles are probably not affected greatly by the relatively low sucrose concentration gradients and short centrifugation times in rate-zone ultracentrifugation, isopycniczone ultracentrifugation in high sucrose concentration gradients is not recommended for determining buoyant density properties of casein micelles in their native state of dispersion. References (1) Anderson, N. G. 1966. An introduction to particle separations in zonal centrifuges, p. 1-40. In National Cancer Institute Monograph No. 21. National Cancer Institute, Bethesda, Maryland. (2) Beckman Technical Review 1. 1960. An introduction to density gradient centrifugation. Beckman Instruments, Inc., Palo Alto, California. (3) Decelles, G. A. 1967. Investigation of the caseinate-phosphate calcium complexes as they exist naturally in milk. Ph.D. Thesis. Iowa State Univ., Ames. (4) Ford, T. F., G. A. Ramsdell and T. G. Alexander. 1959. Apparent specific volume of the calcimn caseinate-calcimn phosphate complex in milk. J. Dairy Sci., 42: 397.

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(5) Handbook of Biochemistry. 1968. Chemical Rubber Co., Cleveland, Ohio. (6) Jenness, R., and J. Koops. 1962. Preparation and properties of a salt solution which simulates milk ultra~iltrate. Netherlands Milk Dairy J., 16: 153. (7) Jenness, R., C. V. Morr, and R. V. 5osephsou. 1966. Some properties of colloidal phosphate-free skimmilk. J. Dairy Sci., 49: 712. (8) Josephson, R. V. 1970. Fractionation and characterization studies of casein micelles and their subunits in milk. Ph.D. Thesis. University of Minnesota, St. Paul. (9) Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265. (10) Martin, R. C., and B. N. Ames. 1961. A method for determining the sedimentation behavior of enzymes: Application to protein mixtures. J. Biol. Chem., 236: 1372. (11) Morr, C. V. 1967. Effect of oxalate and urea upon ultracentrifugation properties of raw and heated skimmilk casein micelles, J. Dairy Sci., 50: 1744. (12) Morr, C. V. 1971. Comparison of protein preparation procedures and starch versus polyacry]amide gel electrophoresis for examining casein degradation products in cheese. J. Dairy Sci., 54: 339. (13) Morr, C. V. and R. V. Josephson. 1968. Fractionation of skimmilk casein micelles by Sepharose chromatography and sucrose gradient centrifugation. J. Dairy Sci., 51 : 943. (14) Ribadeau Dumas, B. and J. Garnier. 1970. Structure of the casein micelle. The a~cessibility of the subunits to various reagents. J. Dairy Res., 37:269. (15) Rose, D. 1969. A proposed model of micelle structure in bovine milk. Dairy Sci. Abstr., 31 : 171. (16) Rose, D., D. T. Davies, and M. Yaguehi. 1969. Quantitative determination of the major components of casein mixtures by column chromatography on DEAE-cellulose. J. Dairy Sci., 52: 8. (17) Tessier, H., M. Yaguchi, and D. Rose. 1969. Zonal ultracentrifugation of fl-laetoglobulin and K-casein complexes induced by heat. J. Dairy Sci., 52:139. (18) Thompson, M. P. 1969. Private communication. (19) Thompson, M. P., R. T. Boswell, Virginia Martin, ]~. Jenness, and C. A. Kiddy. 1969. Casein-pellet-solvation and heat stability of individual cow's milk. J. Dairy Sci., 52: 796. (20) Whitnah, C. H., and W. D. Rutz. 1959. Some physical properties of milk. VI. The voluminosity of caseinate complex in milk and reconstituted sediments. J. Dairy Sci., 42 : 227.