MOLECULAR WEIGHT STUDIES ON ACID - PRECIPITATED, CALCIUM . PRECIPITATE), ARI-IA” M9 BETA CASEINS BY OSMOTIQ PRESSURE MEASUREMENT IN 6.66 M UREA Thesis for I‘I'ze Degree of 95. D. MICHIGAII SMTE UNWERSETY Harald Christian Nielsen 1957 IIIIIIIIIIIIIIIIIIIIIIIIIIIIII I 31293010897324 ‘ If L I 8 RA R Y Michigan State Umvcruty MICHIGAN STATE UNIVERSITY EAST LANSING, MICHIGAN PLACE ll RETURN BOX to remove this checkout from you! ncord. TO AVOID FINES Mum on or before date duo. DATE DUE DATE DUE DATE DUE -I__f—I I—‘I—JI I MSU IoAnNflrdevo Mon/Equal 0M "mm lion Mani-M NOLECULAR NEIGNT STUDIES on ACID-PRECIPITkTFD, CAICIUM- PR CIPIT TED, ALPHw, AND BETA CASEINS BY osmOTIC PRESSURE MEASUREMENT IN 6.66 m sta by Harald Christian Nielsen A TIIES 3 Submitted to the School of Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOTH‘ Department of Chemistry 1957 A. C I‘ZNOL’.’ LEDGL'I'I: HITS The author wishes to express thanks to Dr. E. A. Lillevik for his patience, interest, and encouragement during the course of this investigation. Grateful acknowledgement is due other members of the chemistry department for information as well as the use of various items of equipment. The cooperation of the ser- vice staff is also appreciated. In addition, the writer wishes to thank the American Dairy Association for financial support during the last part of the study. Finally, he wishes to acknowledge the help and en- couragement given by his wife. ii VITA The author was born April 18, 1950, in Chicago, Ill- inois. He received his elementary school education in Oak Park, Illinois, and graduated from Oak Park Township High School in 1948. He entered St. Olaf College, Northfield, Minnesota, in 1948, majored in chemistry, minored in bio- logy and mathematics,and graduated with.a Bachelor of Arts Degree in 1952. He entered the School of Graduate Studies, Hichigan State University51n 1952 and since has majored in biochemistry and minored in organic and analytical chemistry. He was an assistant in the Research Division, Armour and Company, Chicago, Illinois, during the summers 1951 and 1952; a Graduate Teaching Assistant in the Department of Chemistry, Michigan State University, from 1952 until 1957; and a Special Graduate Research Assistant during the Spring Quarter of 1957. He will commence employment at the North- ern Regional Research and Development Division, United States Department of Agriculture, Peoria, Illinois, in July of 1957. He is a member of the American Chemical Society, and its Division of Biological Chemistry; Sigma Xi; and Alpha Chi Sigma. Se is married and has one daughter; iii ti;1m MOLECULAR HEIGHT STUDIES ON ACID-PRECIPITATED, CALCIUH- PRECIPITRTED, ALPHA, AND BETA CASEINS BY OSLOiIC RESSURE MTASUREMBNT IN 6.66 M UREA by Harald Christian Nielsen AN ABSTRACT Submitted to the School of 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 Chemistry 'Year 1957 C‘ k -- 9......“ g; .r f / / _- /,I ‘ ~ \ Approved ‘::2;é%§z;:7<;%ézggf%<§: -O‘ — 30-“ # ABSTRACT The osmotic pressure behavior of acid-precipitated, calcium-precipitated (1), alpha, and beta caseins in 8.66 K urea buffered to pH 4.8 or 8.0 with acetate of ionic strength of 0.1 was studied in order to determine their molecular weight values. Alpha and beta easeins were prepared by fractional pre— cipitation from strong urea solutions according to a modifica- tion of the procedure of Kipp gt,‘§;.(2). During fraction- ation, urea concentrations were determined by measurement of refractive index after dcproteinisation with trichloracetic acid. Osmotic pressure measurements were made at 10°, 20°, and 50° C. using both the Bull and the Fuoss-Mead osmometers; the latter was modified in order to permit the use of toluene as a manometer fluid. Solutions were analyzed for casein content before and after osmotic pressure measurement by determining their absorbence at 2802ma. The equilibrium osmotic pressure (P in cm. H20) for each measurement was arrived at by examination of a plot of os- ‘motic pressure versus time. The equilibrium osmotic pressure was converted to its 0° value (Po) and then divided by the corresponding protein concentration (C in gm./100 ml.) to obtain the reduced osmotic pressure (Po/C). The results for V each casein preparation were expressed in a linear plot of Po/C versus C whose equation was determined by the method of least squares. Molecular weight (N) values were calculated using the van't Hoff equation in the following form: (29 = 52 C M lim. 0:0 in which T is the absolute temperature, and R the gas con- stant (848 cm. H20 - 100 ml. per gram per degree). The results are summarized in the following table: TABLE I MOLECULAR HEIGHTS AND PO/C VERSUS c EQUATIONS FOR case IS DISSOLVED IN 6.66 M UREA Casein Molecular Po/C vs. 0 Mess. Temp. . Preparation Weight Equation pH (Deg. C.) Acid- 28,700 Po/C = 8.08-+ 0.5990 -4.e 50 Precipitated Calciumr 29,800 PO/C = 7.78 + 0.6010 6.0 10 Precipitated Alpha 27,800 Po/C =-o.34 —-0.2470 4.8 10,20,30 Beta 25,100 PO/C =10.01 4-0.6060 4.8 10,30 Calcium-precipitated casein was studied at pH 6.0 because during its isolation the protein was never exposed to hydrogen ion concentrations greater than found in skim milk. It had been proposed (1) that the high solubility and simple sedimentation pattern of this protein at neutral pH vi was a result of such treatment. The other caseins were examined at pH values near their isoelectric points. The molecular weight values expressed for acid-precip- itated and calciumrprecipitated cas eins are number averages, because these proteins are mixtures and osmotic pressure is a function of the number of molecules per unit volume. The nearly identical molecular weight values given by acid- precipitated an ad Cl‘CLUP-DPOC. itated caseins (28, 700 and 29,800 respectively) plus the very similar electrophoretic patterns they produce indicate a close resemblence between these proteins. The negative slope observed in the Po/C versus C plot for alpha casein suggests the possibility of aggregation with increasing protein concentration. Molecular weight was calculated from extropolation to zero protein concentra- tion where this effect would be most diminished. Since concentrated urea solutions are regarded to have a strong deaggregatir .3 effect on proteins, the molecular weight of 27,800 for alpha casein as determined by this no cthod is considered minimal value. The value of 25,100 found for beta casein is also con- sidered to be minimal again due to the deaggregating effect of urea. An axial ratio of 8.2 was estimated for this protein by substituting into the equation (5): P VCRT _1_ ‘s’ Jrv-“a' vii in'which.v'is the partial specific volume and l/d is the axial ratio. In this calculation it is ass ted that the lepe of the Po/C versus C plot is due only to asymmetry of the protein molecules. 1. F. von Hipfiel and D. J. Naugh. J. Am. Chem. Soc., 31, 4311 (1955). - 2. N. J. Hipp, M. L. Groves, T. L. Kcfiockin. J. Dairy Sci., .722: 272 (1952); 5. J. T. Edsall. In "The Proteins", H. Neurath and K. Bailey, eds., Academic Press, Inc., New York, N.‘YM,1954, V01. 14 p0 5950 viii TABLE OF CONTENTS Page I. INTRODUCTION. . . . . . . . . . . . . . . . . . 1 II. EIISTCRICILL o o o o o o o o o o o o o o O o o o o 2 A. Casein and its Fractions. . . . . . . . . . 2 B. Molecular Weight Studies . . . . . . . . . . 8 1. Studies on Acid-Precipitated Casein.. . 8 2. Studies on the Components of Casein.... 10 C. Measurement of Osmotic Pressure . . . . . . 14 III . EiPERIIEEIIrl-‘l‘tll o o o o o o o o o e o o o e o o o 0 17 A. Apparatus . . . . . . . . . . . . . . . . . 17 B. Reagents, Materials and Analytical Procedures . . . . . . . . . . . . . . . . . 22 1. Reagents and Materials . . . . . . . . 22 2. Analytical Procedures . . . . . . . . . 28 C. Experimental Procedures . . . . . . - . . . 3o IV. DISCUSSIOII [XII-D COIICLUESIC’IZS o o o o o o o o o o o 48 A. Heasuremcnt of Osmotic Pressure . . - . . . 48 O U! G) . Casein Preparation and Fractionation Analytical Procedures . . . . . . . . . . . 60 B C D Results of Osmotic Pressure Keasurement . . 62 l. Acid-Precipitated Casein - . . . . - . 2 2. Calcium-Precipitated Casein . . . . . . 65 5. Alpha, Casein o o o o o o o o o o o e o 66 4. Beta Casein . . . . . - - . - - . - 67 V. 8 £13.: [L IZY o o o o o o o o o o o o o o o o o o o o 70 VI . BIBLIC’GPL/‘117I'IY o o o o e o o o e o o o o e o o o 72 VII 9 AP PLJITDI}; I o o o o o o o o o o o o o o o o o O 80 VIII. APPEJDIKIIoococo-coo--~-----lll Table Page I. A Comparison of Components Isolated from Casein by Various Workers . . . . . . . . . . 7 II. A Comparison of Molecular Weight Values Obtained'for Casein.end its Two Main Components by Different Experimental Methods . . . . . . . . . . . . . . . . . ()1 N) III. Absorbancy Indexes of the Caseins . , . , , , IV. Osmotic Pressure Measurements on Acid- Precipitated Casein in 6.66 M Urea Buffered to pH 4.8 with Acetate Ionic Strength 0.1 . . . . . . . . . . . . . . . . 44 V. Osmotic Pressure Measurements on Calcium! Precipitated Casein in 6.66 M Urea Buffered to pH 6.0 with Acetate Ionic Strength in 6.661.§Urea................. 45 VI. Osmotic Pressure Measurements on Alpha Casein in 6.66 M Urea Buffered to pH 4.8 with.Acetate Ionic Strength O.l , , , 46 VII. Osmotic Pressure Neasurcments on Beta Casein in 6.66 N Urea Buffered to pH 4.8 with Acetate Ionic Strength 0.1 . . , , 47 ~- 41. LIST OF FIGURES Figure Page 1. The Bull Osmometer . . . . . . . . . . . . . . . l8 2. The Fuoss-Mead Osmometer . . . . . . . . . . . . 19 5. ElectrOphoretic Patterns of Acid-Precipitated and Calcium-Precipitated Casein Preparations . . 23 4. ElectrOphoretic Patterns for.A1pha and Beta Casein Preparations. . . . . . . . . . . . . . . 26 5. Absorbance Versus Concentration Plot for Acid- Precipitatcd Casein in 6.66 M'Urca . . . . . . . 30 6. Ultraviolet SpectrelTransmission Curves. . . . . 31 7. Refractive Index Versus Origional Urea Concen— tration for Strong Urea and Casein-Containing Urea Qolutions after Addition of an Equal Volume of 20% Trickloraqetic Acid . . . . . . . . . . . 34 8. Electrophoretic Patterns of Acid-Precipitated and Calcium-Precipitated Caseins after Measure- ment of Osmotic Pressure . . . . . . . . . . . . 55 9. Electrophoretic Patterns of Aiph and Beta Casein: after Hessuroment of Osmotic Pressure, , 56 10. Plot of.Reduced Osmotic Pressure Versus Concent- ration for Acid-Precipitated and CalciumrPrecip- itated Caseins in 6.66 M Urea. . . . . . . . . . 39 11. Plot of Reduced Osmotic Pressure Versus Concent- ration for Alpha and Beta Cnseins in 6.66 m Urea 4O 12. Plot of Reduced Osmotic Pressure Versus Concentra- tion for Data of Burk and Greenbcrg on.Acid- Precipitated Casein in 6.66 M Urea . . . . . . . 64 xi I. INTRODUCTION In 1930 Burk and Greenberg (1) reported the molecular weight of casein to be 33,600 by osmotic pressure measure— ment in 6.66 M urea solutions. This value was quite low in comparison with results obtained by other methods. Casein has since been shown to be a mixture of at least three proteins. A number of chemical and physico-chemical investigations have been conducted on its two major com- ponents, alpha and beta caseins. However, the minimal mole- cular weights of these components have not as yet been un- eduivocally determined and no molecular weight determin- ations have been reported on alpha and beta caseins in strong urea solutions. On this basis it was decided to investigate the osmotic behavior of alpha and beta caseins in 6.66 H urea solutions in order to determine the minimal molecular weights exhibited by these proteins in this medium. During the course of the investigation a procedure for preparing casein by adding calcium ion to skim milk rather than adding acid was reported (2,3). It was further decided to compare the osmotic behavior of acid-precipitated and calciumrprecipitated caseins in 6.66M urea solutions. II. HISTORICAL A. Casein and its Fractions Casein is the phosphorus-containing protein which precipitates from the milk of various animals upon addition of acid. Most studies on casein have been on the product obtained from cow's milk and the term casein usually refers to bovine casein unless otherwise specified. Casein was first isolated in relatively pure form by mulder (4) in.1838 by adding acid to skim milk; this method is still the most common way of preparing the protein. To- -ward the end of the nineteenth century Hammarstert (5,6) at the Upsala University, Sweden, conducted extensive chemical and solubility studies on casein and established the belief that it was a homogeneous protein, a belief which.was dom- inant for a long time. In 1918 Osborne and Wakeman (7) isolated from alcoholic washings of casein small amounts of a protein which was low in phosphorus. However this new protein was merely consid- ered a contaminant. Results of solubility studies by Linderstrflm-Lang and Kodama (8) at the Carlsberg Laboratory in 1925 cast serious doubt upon the idea that casein was a homogeneous protein. By extracting casein with dilute hydrochloric acid they were able to separate it into 3 fractions that differed in solubility and in phosphorus to nitrogen ratio. In 1929 Linderstrflm-Lang (9) described the separation of casein into a series of seven fractions. This was accomplished by extracting casein with 60% ethanol acidified with hydrochloric acid and precipitating the protein from the extracts with sodium hydroxide. His frac- tions showed differences in amino acid composition, solubil- ity, phosphorus to nitrogen ratio, coagulability'with rennin, and several other properties. _ In 1934 Groh gt. 5;. (10) developed three methods for fractionating casein: 1) fractional precipitation from 6.66 M urea by addition of ethanol, 2) fractional precipitation from anhydrous phenol at 70° C. by addition of ethanol, and 3) fractional precipitation by addition of hydrochloric acid to a 70% ethanol extract of casein containing ammonium hydroxide. In 1933 Cherbuliez and Schneider at Geneva (11) fractionated casein by extracting it with solutions of neutral salts such as: ‘magnesium.su1fate, sodium chloride, ammonium.sulfate and amtonium.chloride. In the light of recent work, the only homegeneeus protein obtained by these earlier workrrs is the alcohol-soluble protein reported in 1918 by Osborne and flakeman which is novicalled gamma casein (12). Ultracentrifugal studies on casein in.M/30 phosphate buffer at pH 6.8 by Svedb>rg and Carpenter (13,14) at Upsala in 1930 also indicated that casein is not homo- geneous. The number and sedimentation rates of the comp ponents was found hinhly dependent upon the history of the sample. In 1939 Melander (15) demonstrated by Tiselius moving boundry electrOphoresis at pH values near neutrality, the presence of three components in casein which he designated alpha, beta, and gamma in decreasing order of electrophoretic mObility. In 1944 Warner (18) from the Eastern Regional Laboratory, described a method for separating alpha and beta caseins based on the relative insolubility of alpha casein at pH 4.4 and 2° C. in aqueous solutions. In 1950 Kipp 23. El. (12) from the same laboratory announced a procedure for isolating gamma casein based on its relative solubility in 50% ethanol solution containing ammonium acetate at room temperature and apparent pH 4.5. In 1951 Hipp 23.‘gl. (17) patented a procedure for isolating alpha casein which de- pended on its relative insolubility in 50% ethanol contain- ing ammonium acetate at apparent pH 6.5. In 1952 the same workers (18) published two procedures for separating casein into its alpha, beta, and gamma components: one based on fractional precipitation by addition of acid to 503 ethanol; the other based on fractional precipitation by addition of water to casein dissolved in 6.66 M urea at pH 4.6 and room temperature. As dilution proceeded alpha casein precipitated first and.beta next. They were granted a patent on their urea procedure in 1955 (19). In 1959 von Tevel and Signer (20) described a procedure for separating alpha and beta caseins by countercurrent distribution in a phenol-water- ethanol or a phenol-water-glacial acetic acid system. At the present time the urea method appears to be the most practical for separating casein into its alpha, beta, and gmmaa fractions, Under'most experimental conditions, the components of acid-precipitated casein are alpha, beta, and gamma. How- ever, in 1944 Warner (16) showed that alpha and beta caseins were not electrophoretically homogeneous at all pH values, especially those below their isoelectric points. Cherbuliez and Baudet (21) in 1959 isolated another component of casein by virtue of its solubility in 10% trichloracetic acid; they called this component delta casein. In 1950 they (22) des- cribed the further separation of alpha casein into two sub- fractions on the basis of solubility in 5% ammonium sulfate at pH 6 and 40° C. These workers designated the soluble portion as alpha-l casein and the insoluble portion alpha-2 casein,* These two fractions had almost identical phosphorus, tyrosine, and tryptophane contents, In lQSé Waugh applied for a patent (5) on a process for preparing a water-soluble casein, He added calcium ion to “The fractions of casein are designated by Greek letters in most of the literature, e. g. oC-casein, oL,-casein, and 6 —-casein; __—- z.‘ - —-' ” .n._\.:— skim.milk at its normal pH (near 6.6) to fonn large micelles of calcium caseinate that could be removed by centrifugation. Oxalate was used to remove calcium from resuspended calcium caseinate. The resulting casein product was water-soluble up to 50% at neutral pH whereas acid-precipitated casein (its sodium salt) is only about 9% soluble (23). In 1956 Jaugh (24) discovered that reprecipitating calciumeprecipitated casein revealed a new component, low in phosphorus, which he designated kappa casein. He demon- strated that alpha casein dissociated in 0.25 M calcium chloride to yield one part kappa casein to four parts alpha' casein.* Alpha' casein precipitates while kappa casein is soluble under these conditions. McMeekin and co-workers (25) reported at the April 1057 American Chemical Society meeting in Kiami, Florida, that they separated a new component from acid-precipitated casein in 1% yield which they designate alpha-2 casein. When isolated this fraction has an electrophoretic mobility between that of alpha and beta caseins and contains 0.1 — O.l % phosphorus. HCHeekin's alpha-2 casein and Jaugh's kappa casein, seem similar in some respects. Further work:may prove those “Waugh calls this protein alpha casein (2,24). Since it is not the same protein that other workers (15,18,22) call alpha casein, it will be referred to as alpha' casein in this thesis. TABLE I A courialsoa 0F conFeNENTs ISOLATED FROM CASUIN BY VARIOUS WORKE t?a} Component flP Sedimentation Diffusion Electro- Isoeleo- Constant Constant phoretic tric pH 7 pH 12 x 107 dobility pa Alpha 0.99“ 6.5 s 2.908 -s.7 T° 4.54’ 5.992 . a, h 0 0‘. Beta 0.61 1.3b 1.14” 6.05: ~o.l 4.9 1.57e 7.11 Gamma 0.11“ «"0" 6.0° Kappa 0.5” 13.35 1.3f’ orless Alpha' 1.14b 7.11b Alpha-2 0.1.c -5.oc 5.8—6.0c MeMeekih 0.15 Alpha-1 1.1‘ .7.84 Chet-bad- €2- qmi qud ct Alpha-2 1.0% -7.6( Chet-belie: «v.4 Baudet Notes on Table I. a. Gordon et. al.(27,28,° 28). conte at 00 c. McMeekin 6t. a1.(25, 34). They find that the nitronen nts fiof alpha, beta, and gamma cas sins are 15.53%, 15. 55p, and 15.40% respectively. pf'lq b. Waugh and von Hippel (2, 24). C. a They ran their sedimentation studies in phosob ate buffer ionic strength 0.19 at 0°C. Their diffusion studies were run at pH 11.5 in a potassium chloride, potassium.hydroxide system ionic strength 0.15, They make their electrophoretic analyses in veronal buffer pH 8.4, ionic strength 0.1 with 0.05 I5 (Ev-t d. Cherbuliez and Baudet (22) e. Sullivan gt, a1. and d 33 (35). iffusion sodium chloride added. They made their electrophoretic analyses in veronal buffer pH 7.8, ionic strength 0.1. strength 0.1 at temperatures below 15° 0. They conducted their sedimentation studies in veronal buffer pH 7.8, ionic C3 proteins identical. Table I compares the properties of the components of casein isolated to date. At the present time no definite conclusions can be made concerning the absolute number of components of casein except to say that for a wide wariety of conditions its two major components are alpha and beta caseins. B. holecular Jeinht Studies 9n the Cascins 1. Studies on acid-precipitated casein Early workers calculated.minima1 molecular weight values for casein from analysis for elements, amino acid content, and base-combining capacity. Cohn 23'.El¥ (26), after re- viewing the literature previous to 1925, concluded that the minimal molecular weight of casein is about 192,000. How- ever, more recent determinations by Gordon'g§.Igl, (27,28, 29) of elements and amino acids present in casein indicate that the values obtained by early workers were too high. In 1930 Burk and Greenberg (1) determined the molecular weight of casein to be 33,600 by osmotic pressure measure- ment in 6.66 M.urea buffered to pH 4.6 with acetate. In 1936 Bilenshii and Kastorakaya (30) also by osmometry determined the molecular weight of casein when dissolved in anhydrous phenol; they reported a value of 25,000. Svedberg and Carpenter (13,14) in 1930 ran sedimentation analyses on casein in M/30 phosphate buffer at pH 6.8. They found casein to be polydisperse and calculated, on the assumption of spherical particles, that most of it had a particle weight ranaing from 75,000 to 100,000. Extrac- tion of casein with hot 705 ethanol gave a product which was monodisperse in the ultracentrifuge and showed particle weight of 575,000. In 1056 Pederson (51) also made sedimen- tation analyses on casein in phosphate buffer as well as in decalcified skim.milk. He concluded that casein had at least six components and that the particle size of each increased with protein concentration. In skim milk casein exists in association with calcium and some phosphate ions as relatively large particles re- ferred to as calcium caseinate micelles. Their light scattering is responsible for the white color of skim.mi1k. By sedimentation and viscosity studies on skim.milk Nicola gt.'gl.(52) in 1951 concluded that the calcium.caseinate micelles are roughly spherical with a diameter mean of 900 K and range of 80-2000 K. Hostettler (35) in 1954 by use of the electron microscope found the average diameter of skim milk calcium caseinate to be 1000 K. The size of these micelles is quite dependent on pH and calcium ion concentra- tion. As the pH of skim milk is lowered the net charge on the particles decreases. This decrease in net charpe allows aggregation as the isoelectric point is approached. Micelle size also increases upon addition of calcium.ion since calcium.tends to bind casein molecules together. lO 2. Studies on the components of casein In a review published in 1954, McMeekin (54) stated that no molecular weight studies had been made on the sep- arated electrophoretic components of casein. Since then several investigations have been reported. In 1955 Sullivan 33, 3;. (55) reported on the sedimenta— tion and diffusion behavior of alpha and beta caseins. Alpha casein had a molecular weight of 121,800 in veronal buffer pH 7.78 and ionic strenpth 0.1. Although the sedi- mentation pattern for alpha casein under these conditions was single-peaked, the diffusion curve indicated some heteroaeneity. Beta casein had a molecular weight of 24,100 in the same buffer at temperatures below 150 C. At higher temperatures a higher molecular weight component appeared in the sedimentation pattern. In phosphate buffer pH 7.0, ionic strength 0.1, alpha casein showed a complex sedimenta- tion pattern whereas beta casein behaved the same way it did in veronal buffer. HeMeekin and Peterson (56) confirmed the results of Sullivan and co-workers with the further finding that the sedimentation constant of alpha casein was highly dependent on ionic strength. The §20 value of alpha casein increased from 1.0 to 5.7 as the ionic strength.was increased from 0.01 to 0.20 (buffer not specified). (From McMeekin's §20 value of 1.0 and other data from Sullivan 33, 3;. (35) a.mo1ecular weight of 50,500 can be estimated for alpha casein.) ll Sedimentation analyses by Luck and Joubert (57) showed that the §20 value of alpha casein was quite sensitive to conditions of preparation and subsequent treatment whereas the §20 value of beta casein was not. Sedimentation and diffusion analyses were made by von Hippel and Waugh (2) on calciumrprecipitated casein at various temperatures and pH values above pH 7. 'Under most conditions they found two sedimentating components; they attributed the faster component to alpha casein (in reality alpha' casein) and the slower to beta casein. At temper- atures near 00 C. and at pH 12 in a buffer containing potassium.chloride, potassium oxalate, and potassium hydrox- ide of ionic strength 0.25, calciumrprecipitated casein exhibited its minimum molecular weight. The entire sample sedimented as a single component of molecular weight of 15,000. Further analysis led them to conclude that the molecular weight of alpha casein (in reality alpha} casein) was 15,000 -15,000 and that of beta casein was 15,000 - 25,000. Later Waugh (24)reported that the sedimentation constant of kappa casein at pH 12 and temperatures near zero was very close to that of calciumpprecipitated casein under the same conditions. The §20 values were 1.27 for kappa casein and 1.18 for calcium-precipitated casein. Light scattering studies on alpha and beta caseins were conducted in 1952 by Halwer (58) in potassium.chloride, V) ‘J sodium.hydroxide systems at pH 7 and 10. He observed for both alpha and beta caseins an increase in light scatter- ing, especially at pH 7, with increase in salt concentration; he interpreted this increase in light scattering as being due to aggregation. Extrapolation of light scattering values to zero salt concentration and zero protein concentration led to a range of 25,000 to 65,000 for the molecular weight of alpha casein. D'yachenko and Ylodavets (59) reported a value of 52,000 for the molecular weight of alpha casein obtained by light scattering measurements at pH 9 (potassium hydroxide). Quantitative determination of N-terminal groups has also been.used to estimate molecular weight values for alpha and beta caseins. Hoover gt, 31, (40) reported the average chain weight as 10,000 for alpha and 14,500 for beta casein. Mellon gt..gl. (41) stated that.a1pha casein contained 10.7 moles of N-terminal argininc and 1.6 moles of Néterminal lysine per 100,000 grams of protein for a chain weight value of 8,200 grams per H-terminal group, and beta casein contain- ed 5.5 moles of N—terminal arginine and 2.4 moles of H-terminal lysine per 100,000 grams protein for a chain weight value of 15,000 per N-terminal group. In 1857 Wissmann and Hitschman (42) reported 1.4 moles of N-terminal lysine per 100,000 grams of alpha casein; this value agreed well with that of Mellon 23. 21, However, Wlssmann and TABLE_II A COMPARISON OF IOLUCULXR JEIGHT VALUdS OB TilfijD FCR CASBIH AND ITS TWO MAIN CCEPONENTS BY DIFFERENT EXPERIKEfiTAL METHODS Acid- Calcium Alpha Beta MethOd Precipitated Precipitated Casein Casein Casein Casein Element and Amino Acid 192,000q 51,000“ 28,700b Analysis Osmotic 28, 700 ' n a Pressure 55,6002’ 29,800c 27,eooc 25,100 Measurement 25, 000 h “edimenta4-~ 75, 000- 121,800, tion Analy- 100, 000‘ 15,000‘J 50,500‘ 24,100h sis Light 25, 0007 s a 65,000 scatter_ng 52 cook I End Group 10,000 14,300’m Analysis 8, 200M 15, 000"1 51, 000" Hotes on table II. .— e. Cohn et. a1. (26). ‘ b. Calculatedffrom data of Gordon et. a1. (27 29). A value of 29, 600 can be calculatSd for gamma ' casein from.their data. c. This work, in 6. 6 M urea. d. Burl: and Greenberg (l) in 6. 66 M urea. e. Bilenshii (50) in anhydrous phenol. f. Svedberg and Carpenter (15) in phosphate buffer, neutral pH. ‘ ,. Vbn Hippel and laugh (2), in ph03phate buffer, pH 12. h. Sullivan et. al (55) in veronal buffer, neutral pH. i. 0erived from.data of Sullivan at. al. (55); and McMeckin 36 3 a j. Halwer (as) potassium.chloride, sodium.hydroxide, pH 7 ’ and 10. k.DVYachen1:o and (59), potassium.hydroxide at ’ ' pH 9. 1. Beaver 23. al. (40). m. Melon et. al— (41); x n. Wissmann “E'Nitschman (42). l4 Nitschman only found 1.8 moles of N-terminal arginine per 100,000 grams of protein which yielded a chain weight value of 51,000 grams per mole of N-terminal group for alpha casein. Molecular weight values obtained for the caseins by various experimental methods are compared in Table II. C. Keasurcment of Osmotic Pressure at the present time there does not seem to be any one standard technique for measuring osmotic pressure of protein solutions. The botanist, Pfeffer (45) made the first direct measurement of osmotic pressure in 1877. Using a mercury manometer and a membrane of copper ferrocyanide deposited on a porous clay cup, he measured the osmotic pressure of sucose in aqueous solution as a function of temperature and concentration. Van't Hoff (44) in 1887 observed from Pfeffor's data that an amazing similarity existed between Osmotic properties of solutions and the behavior of ideal gasses. He showed that in dilute solutions the osmotic pressure is equal to the pressure the solute would exert if it were a gas in the volume occupied by the solution. This is the basis for determining molecular weight values from osmotic pressure measurements. The pioneering work on the osmotic behavior of protein solutions was done by Serensen in Denmark and Adair in England. 15 Sorensen.§t, 31. (45) in 1917 made the first careful study of the osmotic behavior of a protein in solution. He determined the osmotic pressure of crystallized egg albumin in ammonium sulfate solutions as a function of protein con- centration, ammonium sulfate concentration, and pH. He demonstrated that the osmotic pressure of a protein solution of given concentration was a characteristic and reproducable quantity which established an important point. Adair (46,47) in 1925 reported on the osmotic behavior of the hemoglobins of several mammals. He was the first to correctly determine that the molecular weight of the mammalian hemoxlobins was in the neighborhood of 67,000. In 1928 and 1929 he published two important papers on the theory of osmotic pressure. In one (48) he stated a general equation for the nonlinear relationship between osmotic pressure and concentration which found wide use; in the other (49) he derived an equation for osmotic pressure on a thermodynamic basis. Molecular weightsof a large number of proteins have been determined by measurement of osmotic pressure. Edsall (50) presented a comprehensive review of the work up to 1954. The studies of Burk andl3reonberg (l) and later Burk (51-55) on the osmotic behavior of proteins in 6.66 M urea are of interest. Comparison of molecular weight Obtained in 3.66 M 1 urea with those obtained in dilute salt solutions shows that strong urea solutions sometimes deaggregate proteins into subunits. 16 Although strong urea solutions are often used to dissolve proteins, the action of urea on proteins is not well under— stood. In a recent review Naugh (56) suggests hat urea ruptures hydrogen bonds and that it forms adducts with protein molecules analogous to the adduets formed between urea and long unbranched hydrocarbons. The osmometers used by earlier workers such as Sfirensen (45), Adair (46), Burk and Greenberg (l),had bag-shaped.membranes which were attached to capillaries. The membrane was simply filled with protein solution, immersed in solvent and allowed to come to equilibrium. The osmometer devised by'Bull (57) in 1941 is probably the most refined of those with a bag-shaped membrane. Develop- ment of osmemeters with rigidly supported membranes made more reproducable measurements possible. The instrument described by Schultz in 1966 (58) and that described by Bourdillon (59) in 1959 are good examples of this develop- Inent. The next development was osmometers with large enough membrane to solution ratios and other modifications that permitted dynamic measurement of approach to equilibrium. Examples of such instruments are those described by Hepp (60) in 1936, Carter and Record (61) in 1939, and Fuoss and Head (62) in 1943. The Fuoss-Head osmometer has found wide pop- ularity for work with synthetic polymers in non-aqueous systems. However there is no record previous to this study of its being used to study proteins in aqueous systems. III. EXPBRIKTHTAL 1x ~Q Apparatus Osmometers. Three Bull osmometers were constructed in the glass shop of Kedzie Chemical Laboratory from an illustra— tion by Lundgren and Hard (63). The Fuoss-Mead osmemeter* i—o f K chiaan State ,3 O "J 0 used was constructed in the machine 3. of Dr. K. B. Goldblum.of General Electric Company. Capil' laries with a 5 m1. chamber at their base were made in tne Kedzie Chemical Laboratory glass Shep so that toluene could be used as a minometer fluid. The osmameters used in this 0 investigation are illustrated in Figures 1 and e. Hembranes for the Bull osmometer were cut from 18/59 inch cellulose casing furnished for this purpose by Dr. C. J. B. Thor of the Viskin; Corporation (Chicago, Illinois). hembranes for the Fuoss—Mead osmemeter were cut from 3.55 inch cellulose tubing purchased from Gen ral Scientific Company (Chicago, Illinois). Before the membranes were used they were boiled three times in distilled water for two hour periods each time and stored under distilled water in a refrigerator as suggested by'Yang and Foster (64). *Appreciation is expressed to Dr. R. L; Guile for the loan of this instrument. 18 } ‘*§::: Solvent 1 Tube Protein Tuee Toluene Manometor Stopcock § l i 3 Membrane | K 1 L‘); Figure l. The Bull Osmometer Capillary Socket . The Fuoss-Mead Osmometer Capillary Capillary Socket Solution Chamber with Bibs to Support Membrane Face View of One of the Half—Cell; [fl §Ldg View of the Assembled Osmometer 20 Temperature resulation. The Bull osmometers were sus- pended in a constant temperature bath with a plate glass front constructed in th- Kedzie Chemical Laboratory machine shop. The temperature was controlled to either 20° or 30° ;I.03° C. A system for circulating constant-temperature water, manufactured by Precision Scientific Company (Chicago, Illinois), was used to circulate water at 10°:t .050 through the jacket of the Fuoss-Mead osmometer. The capillaries were exposed to air at room temperature (25°;f 2°). When either of these constant temperature baths run; Operated at temperatures below room temperature, water cooled by a re- frigerated water bath was circulated through coils in the constant temperature baths. Cathetometer. The instrument was manufactured by Gaertner Scientific Corporation (Chicago, Illinois) and could be easily read to 0.01 cm. and estimated to 0.005 mm. Electrophoretic determination§_were made using the Tiselius Electrophoresis Appa atus Model 58 (Perkin-Elmer Corporation, Norwalk, Connecticut). Conductivity measure- ments were made with the Model RC-IB Conductivity Bridge Industrial Instruments, Incorporated, (Jersey City, New Jersey) using a cell with a constant of 0.4893 (Perkin Elmer, Incorporated). Spectrophotometry. Ultraviolet spectral transmission curves were made on casein solutions with the Beckman DK-2 (T) H Recording Spectrophotometer. Absorbance measurements at 280 mg were made with the Beckman Model DU Spectrophotometer. Refractive Index.was determined with an Abbe type re- fractometer manufactured by the Bausch and Lomb Optical Company (Hew'York, New Yerk). Refractive index.measurements were made at 20° C.; thermostated water was passed over the prism assembly to maintain this temperature. Eflzmeasurements were made with he Beckman Kodel H-Q pH meter equipped with a glass electrode. Dialysis equilibration. An external rotating liquid dialyzer constructed by Djang, Lillevik, and Ball (65) was used. Dialysis equilibration.was also carried out by placing the membrane containing solution in a jar of solvent and shaking it with a Burell Model BB wrist-action shaker. Freeze-drying was carried out using the Virtis Freeze- Dryer (Virtis Company, Yonkers, New‘York); Centrifuges. The International Model 2 centrifuge (International Equipment Company, Boston, Massachusetts), equipped with either a size 267 rotor for 250 ml. bottles or a basket attachment, was used in preparation and fraction- ation of acid-precipitated caseins. The Servall Refrigerated Centrifuge (Ivan Sorval, Incorporated, Norwalk, Connecticut) with a size SS-l rotor for holding 50 ml. stainless steel tubes was used in preparation of calcium-precipitated casein. B. Reagents, Materials and Analytical Procedures 1. Reagents and Materials Chemicals. All chemicals used in the preparation and fractionation of casein, in osmotic pressure measurements, and in analytical procedures were either 0. P. or reagent grade, unless otherwise specified. ggid-precipitated casein. This was prepared from fresh raw skim milk* by a procedure given by Dunn (66), and stored at ~20° C. until used. The air dried product contained» 15.9% moisture and 15.29% nitrogen on a moisture free basis. 'The electrophoretic pattern of the product in veronal buffer (Figure 3) was the same as that obtained by Pipp gt, 3;, (18) under the same conditions. Calciumrprecipitated casein was prepared by the follow- ing procedure adapted from von Hippel and Waugh (2,3). Fifty'ml. of 2 M calcium chloride solution were added with stirring over a period of one hour to one liter of fresh raw skim.milk* at ice bath.temperature. The calcium.caseinate micelles were removed by centrifugation at 14,000 r.p.m. for 90 minutes (25,000 x g) at 0° C. (Servall refrigerated centri- fuge). After the precipitate of calcium.casoinate was slurried in a flaring Blender, 65 ml. of 2 M potassium oxalate was added to solubilize the casein. The resulting *Kindly provided by Dr. J. R. Brunner of the Dairy Department, Michigan State University. ASCENDING DESC EI‘IDING it 7200 at “Q I A.l. , r; \r 1' 1;. v .cc.; ..fa site per lg Isoto_ .A Jlfctrophoretic Pattern: of AciS-Preoipit* (lop) and Calciun-Prccipitatai (eottom) t Pczparation: “ted “ascin in Veronal Euffar pf 3.4, Ionic Strnntth 0.15 (0.1 Veronal and 0.05 Chloridz). 3021111. ‘. 24 precipitate of calcium oxalate was removed by means of the International Centrifuge. The calcium.free casein solution was covered with toluene, pervaporated twenty-four hours at room temperature, then lyophylized. The product, which was stored at ~20° C. is what Waugh (24) tenms first cycle soluble casein and it contains alpha and beta caseins rather than alpha' and beta caseins. The water-soluble product appeared in the form of transparent glass-like flakes and contained 7.175 moisture. Its electrOphoretic pattern is given in Figure 5. glpha and beta caseins. Alpha casein was fractionated from acid—precipitated casein both by the alcohol procedure of Hipp.22.‘gl.(17) and a modification of the urea procedure later described by the same workers (18). Both procedures were carried out at room temperature (25-500 C.). The product prepared by either procedure seemed identical by electrophoretic analysis. Beta casein was obtained from acid-precipitated casein by a.modification of the urea pro- cedure of Hipp 3t._al. (18). The following modification of the urea procedure was used to prepare alpha and beta caseins. One hundred grams of acid precipitated casein and 400 grams of urea (Merck C. P. or Baker C. P.) were dissolved to total volume of one liter. The resultant solution was 6.66 M with respect to urea and 10% with respect to casein. The solution was adjusted to.apparent pH 4.6 (glass electrode) (0 U} by dropwise addition of concentrated ammonium.hydroxide or glacial acetic acid. One liter of water was added dropwise with stirring over a period of about one hour using a sepnsmw~ tory funnel. This diluted the urea concentration to 5.5 M and precipitated crude alpha casein. The gummy'precipitate was removed by centrifugation or simply decanted. The super- natant liquid was adjusted to pH 4.8 and diluted as before to 1.5 M with respect to urea. The crude alpha casein precipitate was redissolved in .750 ml. of 6.66 M urea. (From this point on, urea concentra- tion.must be determined by the refractive index method that will be described, since the casein precipitates occlude a large amount of urea.) The pH was adjusted to 4.6 with either glacial acetic acid or concentrated ammonium hydrox- ide, and water was slowly added until urea concentration was 5.5 M to reprecipitate alpha casein. Alpha casein was dissolved and precipitated once more using 500 ml. of 6.66 M urea. The final alpha precipitate was washed three times with water, three times with acetone, and finally twice with ether after which it was allowed to air dry. The product was stored at -20° C. A Waring Blender was used in the beginning to water disperse the precipitate after which it was recovered by centrifugation. Alpha casein thus obtained contained 2.6%:moisture and in a veronal buffer produced a single-peaked electrophoretic pattern (Figure 4) which was I A ‘ V200 300.; 7.76 Volts per Cm.; 11 Protein # 7‘ ,0 anc.; J. J I lts prr Cm.; 1 Protein Titurc 3. Tltctronhorét'c Patterns of Alpha (Top) and Etta (Wetter) Ca: in Preparations in J rona i C A o . c l Tuf* r p» o Strength 0.15 (0.1 V::onal -'| a 10 aoaiuv Chlorine). -11 and 0.0. F) 1:, similar to that obtained by Kipp 32, 31. (17) under the same conditions. The crude beta casein from the initial fractionation was purified by dissolving in 250 ml. of 4.5 M urea and adjusting the apparent pH to 4.8 (concentrated ammonium hydroxide or glacial acetic acid). By slowly adding water until the urea concentration was 5.0 M, the alpha impurity was precipitated. After removing the alpha impurity by centrifugation beta casein was next precipitated by slowly adding water until the urea concentration was 1.5 M. After a similar reprecipitation, the beta casein product was washed three times with.water, three times with acetone, and twice with ether; then allowed to air dry. The product was stored at -20° C. Again, in this procedure the Waring Blender was used to initially disperse the precipitate in water. The product contained 5.8% moisture and in veronal buffer produced an electrophoretic pattern (Figure 4) like that obtained by Hipp gt, al.(17) under the same conditions. Solvents for osmotic pressure measurements. Four hundred grams of urea (Merck C. P. or Baker C. P.) and 8.2 grams of sodium.acetate were dissolved in water and the apparent pH was adjusted to 4.8 (glass electrode) by drop- wise addition of glacial acetic acid. The solution was then diluted to one liter and filtered through an asbestos pad. This produced 6.66 M urea buffered to pH 4.8 with acetate of ionic strength 0.1. The solvent for calcium-precipitated casein was prepared as just described except that its pH was 5.0. Solutions for osmotic pressure measurement. A sample of air dried casein (acid-precipitated, calciumrprecipitated, alpha, or beta) was weighed into a volumetric flask and dissolved in 6.65 H urea solvent with the aid of a mechanical shaker. The solution was made to volume, filtered, and equil- ibrated against solvent overnight (lo-12 hours) by dialysis. Buffer for_electrophoretic analysis. Veronal buffer pH 8.4 and ionic strength 0.15 (0.1 veronal plus 0.05 sodium.chloride) was prepared by dissolving 5.41 grams of U.S.P. Barbital (5.5 diethylbarbituric acid), 2.95 grams sodium.chloride and 0.1 mole of sodium hydroxide to one liter with distilled water. 2. Analytical Procedures Protein concentration. Concentrations of acid-precip— itated, calcium-precipitated, alpha, and beta casein solu— tions were dete mined both before and after osmotic pressure measurements by the following Spectrophotometric procedure. One ml. of l-Sfl casein solution was volumetrically diluted to 25 ml. (or 50 ml.) with 6.65 M urea solvent. After mixing, the absorbance at 280 mfl'was measured in,a 1 cm. silica cell using a Beckman Kodel DU spectrophotometer. The solvent after osmotic pressure measurements was tested for protein by measuring its absorbence at 280 moq. The protein con- centration and the measured absorbence of the solution are related by the folIOVIing equation, A = a, b c in WhiChlA is the absoroance (formerly called Optical densi 5y), bzis the length of the cell in cm., c,is the protein con- centration of the solution measured in sin ./ liter, and la is the absorbancy index at 280 m4fi The absorbency in- dexes at 280 my in 6.66 In”; urea were experimentally determined for the sane lots of acid-rrecLJitated calciun—precipitated, alpha, and beta case ein 8 upon which osmotic pressvz e measure- ments were conducted. This was accomplished by'measuring the absorbence of solutions or onn concentration (wei 5h out casein sample of known moisture content and dissolve volumetrically) us inc the procedure and equation given above. A plot of absorbence versds concentration (gm./liter) for acid—precipitated casein in a 1 cm. cell is shown in Figure 5. The system obeys the Beer law. ‘Ultraviolet spectral transmission curves for 8.66 M urea solvent and for casein dissolved in 6.66 K urea are given in Figure 6. Table III compares the experimentally determined absorbency indexes of the caseins in 6.68 M urea at pH 4.8 or 6 With absorbency indexes given by Hipp 93. g}; ‘12) WhiCh were determined in water at pH values between 7.2 and 9.4. J— 1 0.005 0.010 C (rn./liter) v. ‘3‘? .i, ‘ W .. . I" . - - -. . ‘ « isure 5. Ansoraonee versus dencxntrwtlon Plot icr O I. ‘. r“ l 9s -: . Ju- 4— ‘ ;~' ,.. ‘ ,. 3‘ 1" ‘ 2 0 Q ‘7 .‘ -'-)-C-l-‘)-?lfl'5C.'hlprint): r1¢ r7}... ln Jo'.‘€’ 1. 93.138. \ a 31 250 250' 500 550 mu Figure 6. Ultraviolet Spectral Transmission Curves for: (a) 6.66 if Urea versus Water, and (b) for 0.0059 gm./liter of Acid-Precipitated Casein Dissolved in 6.66 12:? Urea versus 6.66 LI Urea (b). 32 TABLE III ABSORBAI'TCY INDEXES OF TIE CASEINS in,é;ter in 6.66 M urea Acid-precipitated casein 0.7905 Calcium-precipitated casein 0.6644 Alpha casein 1.02 0.9835 Beta casein 0.475 0.4699 Gamma casein 0.05 In plotting the concentration dependence of osmotic :33?essure, and in calculating molecule weights, protein C> 4- ": A 1. . . 4. .2. ,; . ~ Figure l». IlOb of nuanced Genetic Iressui r: o O ‘3 -. ‘1 .0 -1 A I -0 .0 J. ’ _ ffi' _'_ f noncontrztion Lon acid-Precipitit;a and ““‘hins Di:SOIVEd .I-‘f—‘TALQQK,Q P~,\ agn- .t- .‘n—»‘ L \/.-i..._' J..L.."' .. JCJJJZL toga \qu-".-..- \/ - .-" ;" f‘ Y}. .-. - ’3!) .L _/ a. in M! . \.'J .' t QIO'TJ :43 4O b- _L O l 2 C (gm./10x O 01 - .s o: wl.) Picurc ll. Plot of Lofluc:d Cgmotio Pressure VCPSIS Gone ntration for Alpha and Beta Casein: dissolv>d in 8.f5 M Urea 42 where M is the molecular weight, T is the absolute temper- ature, Po/C is the reduced osmotic pressure, and R is the gas constant which in this case is 848 cm. H20 - 100 ml. per gram.per degree. égigfprecipitated casein. Using the Bull osmometer nine osmotic pressure measurements were made at 30° C. on sel- utions ranging in concentration fromC257 to 2.39 5m./lOO ml. (see Appendix I, pp. 81—89); After statistical consideration, results of six of the measurements were used to determine the molecular weight of acid-precipitated casein. The least squares regression for the reduced osmotic pressure versis concentration plot was Po/C =~8.081-O.399 C (see Figure 10). By substituting into the van't Hoff equation; 8.0O :: 848 X 273 M the molecular weight of acid-precipitated casein is found to be 28,700. This value is plus or minus 400 based on the standard deviation of the intercept. (The calculations involved in determining the least squares regression line and the standard deviation of the intercept are given in Appendix 11:) Calcium-precipitated casein. Three omnotic pressure measurements were made at 10° C. on solutions ranging in concentration from 0.70 to 5.34 gm./100 ml. using the Fuoss-Mead osmometer (see Appendix pp.108-llol. The least squares regression for the reduced osmotic pressure versus [tr-M vuw. ml). a: .‘f! (3 43 concentration plot was PO/C ==7.78-+-0.601 C (see Figure 10). The determined molecular weight is 29,8002L.1100. Alpha casein. Six measurements were made at 50° C. and three more at 20° C. with the Bull osmometer. Two measurements were made with the Fuoss-Mead osmometer at 10° C. and one more at 20° C. (see Appendix pp.7o-95577_,0H0, After statistical considerations eight of the twelve measure- ments were used to determine molecular weight. Concentration ranged from 0.56 to 3.80 gm./100 ml. No difference was observed between.measurements made with either the Bull or the Fuoss-Mead osmometers nor was any temperature effect Observed. The least squares regression for the reduced omaotic pressure versis concentration plot was R/C =:8.34 - 0.247 C (see Figure 11). The determined molecular weight is 27,800 1'. 700. ‘Egtg casein. Three measurements were made with the Bull osmometer at 30° C. and three with the Fuoss-Mead osmos- eter at 100 C. After statistical considerations, five of the six measurements were used in determining molecular weight. Concentrations ranged from 0.55 to 3.00 gm./100 ml. (see Appendix pp.96-98,105-107 ). 'Fhe least squares re- gression for thr ruinCDd osmotic pressure vrrsds concentra- tion plot was: PO/C:=r10,01+'0.SOGC. The deterflinod nolocular weirht is 23,1001:500. Tables IV-VII summarize the r sults obtained for acid pro- cipitated, calcium-precipitated, alpha, and beta caseins at various concentrations. ‘4‘th r L 44 TABLE IV SMOTIC PEESSLURE 1.333113314131313 ON ACID-PRECIEITATED CASE N 1H 6.66 M TREE BUFFERED TO pH 4.8 WITH ACETATE ICNIC STRENGTH.O.1 Cone. Presfi Po/C Temp. Osmometer Mesa. (0%“. (Po"“'"‘9 (Deg. 6. ) Used No.63? 0.37 3.04 8.25 30 Bull 5 0.59 4.91 8.32 50 Bull 5 1.40 12.30 8.80 30 Bull 8 1.41 11.88 8.42 30 Bull 4 1.67 14.76 8.84 30 Bull l 2.39 21.60 9.04 50 Bull 7 Po/C = a.oe+ 0.3990 M: 28,700 :1; 400 *The observed equilibrium osmotic pressure is converted to its 0° C. value (PC). **Data obtained in determination of equilibrium osmotic pressure for each casein concentration measured is given in Appendix I. 45 TABLE V OSIJOTIC PRESSURE LIE‘SUBEKZIES CIT CALCPJI‘J-I'EIECIPITATED 0.5113311? IN 8.36 37. Uffliifi‘; BUFFEZLD TO pH 6.0 JITZ: 1-3 1712?”. I’CZTI-fii STIENGTH 0.1 A L _-—_—_ —_——— -— Conc. Presfz' PO/C Temp. Osmometer Meag. ( C 034,) (Po °"“-"‘°) (Deg. C.) Used I~Io."""‘° O . 70 5. 70 8.14 10 Fuoss-Mead 29 l . 77 15. 85 8. 95 ' lO Fuoss-Inlaad 28 5 . 34 52.55 9.74 10 Prose-head 30 130/0: 7.781- 0.5010 1:: :— 29,800 :1: 1100 :"I'he observed eouilibrium osmotic pressure is converted to 11:3 0° C. value (Po). *‘x'Data obtained in detonnination of equilibrium osmotic ~Pressure for each casein concentration measured is given in AID'pendix I. 46 TABLE VI OSKETIC PRESSURE XEASURBEEKTd 0N ALPHA CASE E IN 6.66 M REA BUFFERED T0 pH 4.8 JITH ACETATE IONIC STRENGTH 0.1 Cone. Pres.* Po/C Temp. Osmometer Rees. (c 3%,) (Poe-“4.0) (Deg. 0..) Used No. 0.56 4.60 8.20 50 Bull 15 0.77 6.55 8.48 2. Bull 21 1.68 12.82 7.65 50 Bull 15 1.98 15.40 7.78 20 Fuoss—Kead 22 2.00 14.89 7.45 50 Bull 11 2.51 18.65 8.07 20 Bull 19 3.00 22.79 7.60 50 Bull 10 5.80 28.64 7.54 10 Fuoss-Mead 25 Po/C 8.34 —-O.24VC I: _ l _ 5‘ 27,800 i: 700 * Each equilibrium osmotic pressure value is converted to its 0° c. value (Po). **Data obtained in determination of equilibrium osmotic pressure for each casein concentration.measured is given in Appendix I. OSMOTIC F?L5”U2n MEASUR 47 TABLE VII ELENTS ON BE TA CASEIN 11: 6.6 URF' "UF‘T‘R D T0 pH 4.8 .JITH ACETATE IONIC STRENGTH 0.1 Cone: Pres.* Po/C Temp. Osmometer Meas. (C 3155;}, (Poemmo) (Deg. C. ) Used Ho."z 3' 0.55 5.73 10.66 :50 Bull 18 0.61 6.22 10.20 10 Fuoss-Head 26 0.91 9.52 10.25 50 Bull 17 5.00 55.90 11.97 10 Fuoss-Kead 25 Po/C = 10.01 + 0.6060 1.1 = 23,100 :2: 500 *Each equilibrium osmotic pressure value is converted to its 00 C. value (Po).. **Data obtained in determination of equilibrium.osmotic pressure for each casein concentration measured is given in Appendix I. IV DISCUSSION A33 COWCLESIOVS A. Measurement 2: Osmotic Pressure Bull osmometer. This apparatus has found favor for several reasons: it is completely made of glass (except I. » «4 for :e membrane); it is relatively easy to construct; it p is easy to manipulite; it has a toluene manometer which is more reliable than an aqueouS‘mandectcr; only a small amount of liquid passes across the membrane during a'measurement so it should approach equilibrium rapidly. The apparatus however has its disadvantapes: It's principal disalvantase is probably that the meme brane is not risidly supported. A small shift in theznma- branc during a measurement will produce a large change in the manometer reading and will profoundly disturb the system's approach to equilibrium. Wagner (68) in his ca prehensive review of methods used to measure osmctic pressure published in 1940 refused to discuss devices that do not have rigidly supported membranes. Erratic behavior such.as was observed in the Bull osmoneter could be attributed to shifting o the membrane. Application of gentle air pressure on the protein side to stretch the membrane before starting a measurement reduced its shiftinv. gs ~0 Bubbles in the solutions are also troublesome with the Bull osmcmeter as with other osmometcrs. Bubbles are espec- ‘! O ially troublesome if tney find their Wiy into the toluene manometer. Care must be used when the osmonetor is filled. Application of a partial vacuum to the solution and solvent before placing in the osnoneter nel,ed prevent the formation of bubbles during a measurement. However, some evaporation occurred during this trvatmont which led to unequal urea concentrations in the solutions and solvent and caused or- ratic osmotic behavior during the first few hours of a measurement (see Appendix I p. 94 for an e:-:a1::ple of this.) Trouble with bubbles was also reduced by carrying out the equilibrium dialysis of the protein solution afainst solvent at a temperature equal to or higher than the temperature or measurement. In the hands of its inventor the Bull osmometer would come to equilibrium.overnight (57,69). However,Lundgren and Ward (63) in their detailed directions for using the Bull osmometer suqnost that a measurement be carried out over g.L) a period of at least one week. Eq¢h reasurenent made with a Bull osnometc-r during this investigation I’C!Q1.1il‘0d a per- iod of ton day; or more; thi; was lonrer than the protein solxtion nivht as axe etoi to re sin stable. _'pll 5.7-ab In. A steady decrease in observed osmotic pressure was seen during the latter part of many of the osm tic pressure meas- urements. (See Appendix I, p.85, for an example of this.) Lowering the temperature reduced or eleminatcd this falloff. Reduction in the number of protein particles in the solution as a result ofgpaSsage through the membrane, precipitation, T” absorption onto the membrane, decomposition, or aggregation of the protein might explain the observed falloff. The solvent was shown to be free of protein after meas— urement of osmotic pressure (absorbence at 280 mJL).' There- fore the possibility of protein passing through the membrane could be discounted. The concentration of each protein 501- ution was the same before and after measurement of osmotic pressure (absorbance at 280 mna) which.would indicate that the protein was not precipitating or being absorbed onto the membrane. In each case the electrophoretic pattern of the casein sample was unchanged after measurement of osmotic pres—- sure. (Compare Figures 2 and c; 5 and 9.) This would indicate that the protein was not altered during a measurement. The possibility of protein aggregation during a measurement in 6.66 M urea was neither substantiated nor discounted. The caseins have shown a decided tendency to aggregate in dilute salt solutions, especially at or above room temperature (2,51, 55,56,57,58). Some aggregation in strong urea solution could explain falloff in observed osmotic pressure. Since it was. noted that falloff was reduced by lowering the temperature one could postulate that this factor would reduce aggregation in strong urea solutions. Such a temperature effect upon state of aggregation has been noted in dilute salt solutions (2, 25,37). In this study, the equilibrium.osmotic pressure for a given concentration was determined from a plot of osmotic pressure readings versus time. Allowing the measurement to go until the observed variations were very small could not be used due to the membrane shifting and also falloff in obser- ved osmotic pressure. Approach to a given equilibrium value from both above and below was also tried, but did not work, again due to the membrane shifting and falloff in observed osmotic pressure. (See Appendix I, p.89 for an example.) A plot of osmotic pressure versus the reciprocal of time is linear fer a system asymtotically approaching equilibrium. This type of plct was tried and found not to be linear; this was attributed to the membrane's shifting. The membrane might be considered the most important part of the osmometer. The prime requisite for a.membrane is that it can be semipcreable; that is, that it be imper- meable to the protein but permit rapid diffusion of the solvent. It is also important that the membrane be uniform. Membranes for the Bull osmometer were cut from cellulose tubing furnished by Dr. C. J. B. Thor of the Visking Corpor- ation. Bull and Currie (69) also obtained their membranes from this source. The membranes were quite uniform. In contrast with the eXperiences of Bull and Currie (69); . 14-1! however, the membranes did not permit as rapid ‘iffusion of 0 solvent as might be desired. Haurewitz (70)} s mentioned that the permeability of cellulose tubing can as increased by treatment with aqueous solutions of zinc C1M1041 0; his was not tried. Before using, the membranes were boiled in distilled water as recommended by Yang and Foster (34) in order to remove untriviolet absorbingtznsterial, A common practice his been to cast membranes b7 pouring an ethanol- ether solution of cel ulcse nitrate (cellodion) over a test tube and drying; Alexander and Johnson (67) describe the technique. By altering the cellulose nitrate solution and the time of drying, the pe Leseility can be controlled. The experimental technique is rep rted to be difficult and uni- form membranes are hard to obtain, ['7' ’ Fuoss-Heed Osmemeter, inc membrane erre of tb 113 instru- ment is quite high in relation to the solution volune, and the capillsrics are small. This means ths.t the instrument will come to equilibrium quite rapidly; The Fuoss-Mead osmeraeter hes a membrane that is ricidly sup1mo ted by stain- less steel ribs which eliminates trouble due to membrane Shifting, The Fuoss—Kesd esmometcr was considered better than the Bull osmemetsr; however, it does have its ci.ndvsnt3803; The construction of the instrwnint requires intricste and there efore extensive mac to a 11PCC area of stainless steel which means the metal 53 ion contaminqtion is quite nossible. After a measurement, it was observed that both solvent and protein solutions were yellowish; however, the osmotic pressure usually remained constant during the course of a measurement. In addition to the Cossibilit; of metal ien contamil— ation, the application of the Fuoss-Keid osrcmeter to T)an LI .’" u ”as been limited by the desien .0 i - .1- .. . ns in a ueous sys e s - ,_J of the instrument which uses the solutions beirg Lessured as f manometer fluids in the capillaries. Aqueous solutions, especially urotcin solutions, are poor manometer fluids due to their tendency to stick. The osmometer used in this study was modified so that toluene could be used as a manometer fluid. This worked very well. The presence or absence of bubbles in the Fuoss-Mesd osmomster could not be observed directly, again, due to its metal construction. Solutions were run.back and forth through each of the half cells until it was felt that all bubbles had been removed. Equilibrium.dialysis of the solution and solvent before measurement at the temperature equal to or higher than the temperature of measurement also helped. Fortunately, no trouble was experienced that could be attributed to bubbles. Temperature control with the Fuoss-Mead osmometor is more difficult than with the Bull osmometer. Immersion in a water bath is not recommended because the edge of the mem- brane is exposed which would allow very slow diffusion into (n 95 the water bath. Water at a constant temperature was circul- ated through the jacket of the osmometer; the capillaries were, however, xrosed to air at room temperature. This did cause variations in the readings (see Appendix I, p. 110). It would have been better if the Fuoss-Head osrometer were placed in a constant tourerature air bath. An ideal osm meter fer protein work would have a large rigidly supported membrane; be made out of an inert, trons- parent material; would use small amounts of protein solution; would be easy to manipulate; would have a rapid, sensitive manometer; and would be easy to construct. A Fuoss-Mead osmoneter made out of a transparent elastic would fulfill many of these ideal qualities. Membranes for the Fuoss-head osmometer were cut from cellulose tubinr. These membranes were also boiled in distilled water before using (64) which.rcmevcd some ultra- violet absorbing material. Hith proper choice of membrane a measurement using the Fuoss-head osmoneter can be completed within one hour after filling. The membranes used wore slower by about a factor of about fifty, but they were quite unifbrm. After completion of the measurements it was dis- covered that Carl Schleicher and Schuell Comps y, Keene, New Hampshir sell cellulose membranes suitable for use in he Fuoss-Xead osmometer. One may cast his own membranes from cellulose nitrate (collodion). The difficult tech- nique is descri ed by Fuoss and Head (62). A cellulose U} ‘1 Yifr”;.""7”:1 we '~ . .~.- ‘ ,r ...an ay 3e preferevle to on: O; cellulose nitracL‘ ‘3') l" -. _-' n. 1, r--. r? _‘ n a H llfi . ‘ fO- P- 01.66.11 Jeri ...-Ll’lC".‘ neurozit (7‘9) nan‘tlons tnat the nitro groups on a cellulose nitrate :wwtrmna ~~v sane proteins. Solvent for osmotic pressure measurement. Keasurements were made in 6.66 H urea buffered to app :..rcnt p21 4.8 or 6.0 .W O H: ’7 ' J—l .'—— -s ' ..'-- "‘ Wlofl acetate »nie scren t1 0.1. C. - s - .- s V 1.. 3 "\ so ‘ ‘ . - .- etrone Tree sell.icn Jas cnoson because of its demon- H° strated de a’rre” .t D n9 effect on proteins inclzding the caseins (56,71); the molarity of 6.66 was that used L" '" BUFK and G?C3fihTT8 (1)9 5”fi”’"“il“n and interaction aas ‘Q M q ~ .- _‘ w. I“, : __ ’_ _o .0 ‘ _c_~- _o _ ‘ . .. .‘ J. O proeael; been tne na.or ulfllcalv, in interprething re— sults of molecular Uflifilt determinations on the caseins. ' . . 'L r‘ 4' -‘. w -. l'\ .- . ' 1 more a.o lfidlCQulORS th-t nls uroi solvent sysceu does con? plotely or almost complete 17 eliminate the aggregations and interactions which yield hinr no oloculsr rweight values. ts CL Osmotic Pres ssure Keesurement (p.62). iipp gt. a .(18) showed that the csseins are not rerwonertlr altered by strong urea solutions. Increr a se in alone of the reducei osmotic rressure versus cencent Nation curve is observed when protein molecules in the solution carry a chorée. This occurrs at pH values removed from the isoelectric point of the protein. There- fore, meesurewents on acid—precipitated, alpha, and beta caseins were made at apnarent pH 4.8 which is close to the isoelectric points 5 these proteins. Eencurinentu on calcium-precipitated casein were wade at pH 6.0 because tan soluuility 9nd sedi“3ntntion pattefn of calciut—precinitaicd casein at n utral pH dcpcifi on the fact that Lhis protzin prcpqnation n13 not Eown at or non? its isoslsctric voint. Reducing the pi of a solution of calcium-precipitated ; casein to 4.8 chances it to acid- -pr ecifiitatod cisein. ‘ The c‘fact 0? charge an the protein W'L also rc'ucai hy incr asing t%c ionic ’rncnotn of tne solvent to C.l (sod’um QC‘rtflt'ifl. Ininrirtt’aan an? Tier-d (6.3 sum-«33‘ that an ionic strenmth of 0.1 will r"rootly P3dUC8 the incréaie in observed o-motic p‘esrurfl due to C? “F“ on T‘1"OOtL”‘-J..1'l VOlQCU1C3- B, Casein Pro Wintion a ‘ Fractionation ‘u . 5‘ P . ’. - - ‘l p. n .'.\: fi 4 fl. L1- .. held-greclpltatcd one in J3. pr.pqr,n accoruin co cu r .1 .. - '. ‘rzr: .. L 1-.. _~ L ., '5. ° . procwouro of Dunn (cc). isse_1tially, nfcrocnlonic HClo lb m 0) Ho :3 ‘0 added d;c view to cilufsd skim milk to pricipitata ca ani ,hc pr cipi 3+3 is wgshed with wet r, :thanol, and etEVl cthcr in succession. Io difficulty was oxnerienccd with this oroccdur . Linn ct. L1. (13) sunrist Lhc use of acetone rather tHQn alcohol for final was occause beta .. . .-.?. M . .. .‘ 1.°'1 .1--. L9 .. ,. and "1: n caSains nave anO Loluwilic; in alcohol nni n1- ' A ‘ ‘ I :- ,-. '14s A a L ~2 'l”. r) ilar to t‘ni 0%:ervrd rs a relnlt of law aCLLCH O; r nnln (79). Cncrhulicz nrd 7nudct (?2 report J such a split in 57 one of their casein samples and McMeekin has reported it in casein samples isolated from the milk of individual cows. The split peak was also observed in a commercial sample of casein labeled ”nach Hammarsten" which had been stored at room temperature for several or more years. Casein was therefore freshly prepared by isolation from.mixed herd milk, and after isolation stored at ~20° C, Calcium-precipitated casein was prepared by a procedure adapted from that of Waugh (2,5), It involved: 1) addition of calcium ion to skim.milk to form.micelles Of'calcium caseinatc 2) removal of these micelles by centrifugation, 3) removal of calcium by addition of oxalate which solubilized the casein, 4) dialysis to remove salts, and 5) freeze-drying, Waugh (24) showed that this product is probably identical to acid-precipitated casein which had been dissolved at pH 12 then‘brought to neutral pH. The electrophoretic pattern (veronal buffer) of the product is the same as that of acid- precipitated casein (Figure 5); Waugh (24) discovered that addition of calcium ion to a solution of calciumeprecipitated casein caused its alpha component to dissociate into soluble kappa and insoluble alpha' caseins.* Waugh.removed calcium caseinate micelles from skim milk by centrifugation in the Spinco preparative untracentrifuge at 67,000 x g and 0° C. *wgugh used the termonology, the alpha-kappa complex dis- sociates into alpha and kappa cascins. Other workers (15,18, 22) call the alpha-kappa complex of Waugh (24) alpha casein. - .v' . {it} The same result was achieved in this work by using the Ser- vall refrigerated contrifuge at 25,000 x g and 00 C. How— ever, the International centrifuge run at 1470 x g and 7° C, did not effect separation, £l2§2.22§.§9ta caseins. Alpha casein was prepared by the alcohol procedure of Kipp gt. 5;. (17) and also by a modification of their urea procedure (18). Experience showed that the latter procedure was simpler and gave high- er yields. Beta casein Was also prepared by a modification of the urea procedure of Hipp g§.‘al. (18)‘which.seemed much :more direct than the alcohol procedure presented in the same paper. Alpha casein.was found to be somewhat more soluble in strong urea solutions than indicated by Hipp gt. al. (18). IFor example, the published directions state that at room temperature alpha casein will become insoluble when the urea molarity is reduced to 4,6 by addition of water.- In this study under the same conditions it was observed that alpha casein did not precipitate until the urea molarity was reduced to 3.5. In water solutions.and dilute salt solutions some principal factors that reduce the solubility of alpha casein are: l) adjustment of pH toward pH 4.6 (acid-pre- cipitation), 2) increase in temperature, and 3) addition of calcium ion. I in» *- V. .s 59 In strong urea solutions the minimum solubilities of alpha and beta caseins were found to be at apparent pH values of 4.6 and 4.8 respectively. It was important to maintain the pH very close to these values during fraction- ation in urea solutions. Fractionation in urea solutions was carried out at room temperature (BS-30° 0.). One attempt was made to precipitate crude alpha casein at 5° C. by dilution of a strong urea solution at pH 4.6. However, this fraction did not precipitate until the temperature of the diluted solution was increased to 25° C. The effect of ealcium.or other divalent ion.upon pre- cipitability was not determined. Fractionation was accomp- lished without adding salt. The urea fractionation procedure, although good, re- quires further study.* Quantitative infonmation concerning the factors that affect the solubility of the caseins is required, since variations Observed cannot at present be adequately explained. The possibility of modification in order to separate alpha' and kappa caseins should be con- sidered. A more rapid method for determining relative amounts of the various caseins in a fraction is needed. With electrOphoretic analysis, one has to wait at least twelve hours to find out the composition of a fraction. *Dr. J. R. Brunner, of the Dairy Department, Michigan State University, is currently working on the pilot plant production of casein fractions by the urea procedure. 60 It should also be determined whether variation of tempera- ture and pH, or addition of salts or divalent metals would aid in the fractionation. Although a procedure was available (18), gamma casein Was not isolated due to the small amount present in acid- precipitated casein which makes gamma casein hard to isolate. Kappa casein was reported late in 1956 (24), and no proced- ure is yet available for isolating kappa casein in pure form. C. Analytical Procedures Qasein concentration. In determining molecular weight by measurement of osmotic pressure an accurate method of d‘313¢51r."nainin,r_>: protein concentration is essential. Kjeldahl analysis is the standard method for determin- ing protein concentration; it is often the referee method 11305» to ascertain the accuracy of other methods of deter- mining; protein concentration. After precipitating casein from 8.66 M urea with trichloracetic acid, the semimicro K3°ldam procedure described by Clark ('73) was tried. Diff- iculty was caused by occlusion of urea by the casein pre- c1pitate which caused high results. Adding five volumes of tr"if-”12Loracetic acid reagent in order to dilute the urea m°;a1‘ity, plus washing the precipitate with trichloracctic acid reagent and water did eliminate the difficulty. IYesfractive index has also been used to determine pro- te - in concentration of solutions used in osmometry (69). 61 An attempt to use this method to determine casein concentra- tion in strong urea solutions was without success. The diff- iculty was attributed to small variations in urea concentra- tion. Colorometric procedures are also used to determine protein concentration. A quantativc biuret procedure des- cribed by Gomall 33;. 31;.(74) and used by von Hippel and Waugh was tried (2). This method lacned precision and the Beer law was not obeyed. The method deve10ped where by absorbance at 280 M due to aromatic residues in the proteins was directly measured, was quite accurate and easy to carry out. Small variations in urea and ace ate concentrations did not interfere. Care had to be taken, however, not to get toluene, which was used ‘33 manometer fluid, into the samples upon which a‘vsorbance 1713 St aurements were made. Direct measurement of absorbence at {-390 mat in 0.1 M a"3(1111111 hydroxide, where ionized phenolic residues absorb Strongly, was also found to be quite accurate and conven- lent However, it was considered an advantage to determine protein concentration in the same medium in which osmometry w- . is carried out. Urea concentration. Excellent methods for determining urea are available ('75), but these are designed for 801111310115 containing low concentration of urea. A simple method that c . 0111d be applied directly to strong urea solutions was desired. 62 Measurement of refractive index increment due to urea seemed logical since the property was easy to determine and the method had an appropriate sensitivity. Casein, which was present in varying amounts, unfortunately interfered. .Hevertheless, it was quite easy to remove with trichlorace- 'tic acid, and the result was a linear relationship between the refractive index of the doprotinated solution and its urea cone entration. D. Results _c_>_f_ Osmotic Pressure Measurement 1. Acid-Precipitated Casein Since acid-precipitated casein is a mixture, itsmole- cul ar weight will be an average. Different types of measure- ments yield different molecular weight averages (50,75). Oazrnotic pressure measurement as well as end-group analysis Yield a number average molecular weight (En) which is defined by the equation: En : i miMi i m1 in which mi is the molarity of. an individual component of molecular weight Mi. The weight-average molecular weight (NR?) 13 defined by the equation: Mw = 2’ miMiZ ELI-1‘3. is obtained by sedimentation velocity and diffusion 63 analysis as well as light scattering measurement. The z-average molecular weight (Hg) is defined by the equation: M2 = 5 11111:! 3 zmT'i'Ziii and is obtained by sedimentation equilibrium. For a homo- ‘goneous, monodispcrse substance, these averages are equal to 1? 25,000 by measurement of osmotic pressure in anhydrous IDllenol solution at 50° C. The reduced osmotic pressure versus concentration plot :fWDa? acid-precipitated casein from the data of this investi- Efiettion is given in Figure 10. The corresponding plot from tile data of Burk and Greenberg is given in Figure 12. The 8lopes are identical within experimental error, but there is ‘1 small difference in the intercepts. 1.... Olga C) CD 5.1:. w ~9-“—- Ht " 2 o C (gm./103 ml.) i 1 i 4 0'1 s j 5 rs -: ~fl '. . r1 .* ‘Y ” '\ bone ntrztion ior Data oi _w" and «sch ' . .- . ;-- h J- " ' . '1. .” - e . ‘ Q ‘e e -anuire 3rd. rlrvt o: -1xnioou C1; ot1(:};fiou'tafi VGI‘JIS ‘ f“., ‘ ' .—~~ .9- 1"- . .0 -.-.-,_ ., . vco.n up? (1) on Acid—racoiwit tad ' ..'. “Jr... ‘--- ' .- c- I"? T' 0*- - Gas in ileeOlJ a in 9.30 n Urea. 55 2, Calcium-Precipitated Casein The number-average molecular weight of calciumpprecip- itated casein. measured at 10° C. in 6.66 H urea buffered to pH 6.0 with acetate of ionic strength 0.1 was found to be 29,8001. 1100. This is within experimental error of the ‘value of 28,70011400 Obtained for acid-precipitated casein in the same solvent system. at pH 4.8 and "rteasured at 30° C, {This substantiates the belief that acid-precipitated and calciiun-precipitated caseins are very similar. Other evid- ence for the similarity of the two casein preparations is presented by Waugh and von Hippel (24). We, showed that acid-precipitated casein can be converted to a product that has the sedimentatien pattern and solubility of calcium- precipitated casein at neutral pH by brief treatment at pH 12 and 0° C. This investigation also showed the electro- pl-ioretic patterns of these two caseins to be quite similar (Figure 3). The reduced osmotic pressure versus concentration plots for cilcium-precipitated and acid-precipitated caseins are C=Ompared in Figure 10. The increased slope in the case of calciwi-precipitated over acid-precipitated casein can be e3:1:1ained by the increased charge on the protein molecules at pH 6.0 (isoelectric point near pH 4.8). This difference in slope would have been much greater at a lower ionic 3 t rengtht 66 3. Alpha Casein Waugh and von Hippel (24) have shown that alpha casein can be separated into two compnnents on the basis of solubil- ity in 0.25 M calcium.chloride. However they observed that alpha casein appears homogeneous by sedimentation analysis ,y 5 ”IA. and electrophoretic analysis in phosphate buffer. Other , yvorkers have found alpha casein to be homogeneous in veron- :11 buffer by electrophoretic analysis (18) and sedimentation :xnalysis (35). In this investigation it was observed that sllpha casein has the same single-peaked electrophoretic Pattern both before and after osmotic pressure measurement (I:iguresq37) It is not known whether 6.66 M urea solutions YVlel dissociate alpha casein any further. However, Petersai ( ’71) implies that the sedimentation pattern of alpha casein i;r1 strong urea solution is single-peaked. Also, alpha <=£asein prepared by fractional precipitation from strong 'Ellrea solutions by addition of water is identical to alpha <=€1sein prepared-by other methods (18). The reduced osmotic pressure versus concentration curve 1?C>I‘ alpha casein has a negative slope whereas the curves -fV31? acid—precipitated, calciumrprecipitated, and beta caseins hSLVe positive slopes (see Figures 10 and 11). This may be accounted for by postulating an aggregation of alpha casein which is dependent on protein concentration. This postulate V'Cnlld agree with.the fact that alpha casein is the least ac>lub1e of the caseins in strong urea solutions. 67 The molecular weight determined for alpha casein is 27,800 11700 which is near the value of about 30,500 estimat- ed from sedimentation and diffusion data in veronal buffer of Sullivan 93;; 3;;(35); and Moifieekin and Peterson (36), It also agrees with the value of 51,000 reported by Wissman and Nitschman (42) by end group assay with dinifi‘oflufi'o- laugh (2) indicates that the molecular weight of w benzene. ( alpha’ casein is between 13,000 and 15,000.) The value found in this investigation is also within the range of 25,000 - 65,000 given by Halwer (38) and 32,000 reported by D 'yachenko and Ylodavets (39) both obtained by light Scattering, The agreement of the value obtained in this inVestigation with values obtained by other methods is good evidence that the minimal molecular weight of alpha casein 18 about 30,000; However, molecular weight values for alpha casein given by different experimental methods are not yet precise enough to use as a criterion for the homogenity of 131115 protein as discussed on pageél. 4; Beta Casein The molecular weight value concluded from this investi- £758ition is 23,100 J: 500. This is in good agreement with the Value of 24,100 obtained by Sullivan 93;. 3;. (35) by sedimen- taL‘lsion and diffusion analysis in veronal buffer. It is about t‘vice the value of 14,500 obtained by Mellon _e_:c_. 93;. by end group assay. Von Hippel and Waugh (2) estimate from their data 68» a range of 15,000-25,000 for the molecular weight of beta casein. The close agreement of the results of this investi- gation obtained by osmotic pressure measurement with those of Sullivan 31;. El. (35) obtained by sedimentation velocity can be taken as a criterion for the homogenity of beta Casein (see page 61). The slope of the reduced osmotic pressure versus con- centration curve can be attributed to such factors as: 1) interaction between the protein and solvent molecules vlhich is greater in the case of aseretrical molecules, 2) charge on the protein molecule, and 5) protein aggrega- tion. Edsel (50) gives an equation which can be used to calculate asymmetry from osmotic pressure data assuming that tile slope of the reduced osmotic pressure versds concentra- tion curve is only due to asfiletry of the protein molecules. It is, - RT+ vCRT _l_ ’1'?- 51 d ”i in which l/d is the axial ratio for a rod-shaped molecule and V is the partial specific volume. The experiments in th is study were conducted at the isoelectric point of beta 0233 Sin and with 0.1 It! sodium acetate present which would re duce the effect of charge on the protein molecule to a he Eligible value. Assuming; that the effect due to aggrega- tion is also negligible, it may be estimated from the results of this investigation that the axial ratio or beta casein is 8.2;'that is, the molecule is 8.2 times as long 513 it is wide. Sullivan (55) gives a value of 15.9 for the akial ratio of beta casein from sedimentation-diffusion studies and Hipp _e_1_:_. 3;. ('77) report a value of 13.2 from V1 .3 cos ity measurements. .0. V . $1173!me I. Number—average molecular weight values of r38,700 It 400 and 29,800 I 1100 were determined for acid—precip- itated and calcium-precipitated caseins respectively by osznotic pressure 121 asurement in 6.66 M urea buffered with acetate ionic strength 0.1. 2. IJIinirial molecular weight values of 27,8001: '700 arid 25,100: 500 were determined for the first time by this m3 thod for alpha and beta caseins respectively. These val- ues agree with minimal molecular weights determined for these proteins by other methods. 3. Osmotic pressure behavior and electrophoretic an- alysis indicated that acid-precipitated and calcium-precip- itated caseins are quite similar. 4. An axial ratio of 8.2 for beta casein was calculat- aC1, with certain assumptions, from the slope of its reduced Q'-'3motic pressure versus concentration plot. 5. Tendency toward aggregation with increased concen- tzili‘ation by alnha casein in'6.6 fl 1" o m urea at pH 4.8 was in- C13- cated by the negative slope of its reduced osmotic press- ur‘e versus concentration plot. 6. The method for isolating alpha and beta caseins from acid-precipitated casein by urea fractionation was Inodified, since it was observed that the solubility of '71 alpha casein in strong urea solutions was greater than in- C11 cated by heretofore published procedure of IIipp 913. ill. '7. A method for determining: concentrations of the caseins dissolved in 6.66 M urea based on absorbence at 2.3530m” was developed. 8. A method for determining urea concentration of Strong urea solutions containing casein was worked out. The ma thod was based on the refractive index of the strong urea 3 Olution after deproteinization with trichloracetic acid. 9. The capillaries of the Fuoss-Mead‘osmometer were ITlodii‘ied so that toluene could be used as a manometric fluid. T1113 adapted the instrument to use with aqueous protein sol- 1.11; ions. B-IBLI OGRA PHY girls, 1x1; F., and Greanberg, D. LE. Physical Chemistry of C e Proteins in Non Aqueous and Mixed Solvents. J. Biol. hem” 321: 197-258 (19:50) e. Eon Hippel, P. and Waugh, D. Casein: Monomers and Q ENTIGI’S. J. Am. Chem. Soc., 2'1, 4311-19. égsifiquu D. F. Process for Producing a Water-Soluble again. U. :3. Patent No. 2,744,891, May 9, 1956. 4. . . . . Egiqulder, a, J, Ann. der Pharm., 28, 75 (1838); McMeekin, ., L. Milk Proteins. In "The Proteins", H. Neurath L d_I:Le><:ular i'leight of Certain Proteins. J. Biol. C'hem., 5‘3. 721-55 (1925). garden, a” Semett, 1.7,, Cable, R., Morris, hi2. Amino Acid 1- r7 aposition of Alpha and Beta Caseins. J. Am. Chem. Soc., 4.1L. 5295-7, (1949 . gordon, W3, Semmett, ‘47., Bender, Maurice. Alanine! l‘Jcine, andProline Contents of Casein and Its Compon- ents. J. Am. Chem. Soc., 33, 4282 (1950). fiQrdon, ‘51., Semmett, '31., Bender, Maurice. 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(London , A 109, 292- 500 (1925). Adair, G. S. A Theory of Partial Osmotic Pressures and Membrane Equilibria, with Special Reference to the Application of Dalton's Law to Hemoglobin Solutions in the Presence of Salts. Proc. Roy. Soc. (London), A 120, 575 (1928). lb re 50. 51. 52‘. '76 Adair, G. L. The Thermodynamic Analysis of Observed ESITIQtic Pressures of Protein Salts in Solutions of iii-1‘13- te Concentration. Proc. Roy. Soc. (London), A 1:33, 0-24 (1929). “" Faisal-1' J; To Size, Shape, and Hydration of Protein fl' :3 ecules. In "The Proteins”, H Neurath and 1:. Bailey, ,. s - , Academic Press, Inc. New York, N. ‘1’. vol. _1_, 078-502 (1954). Btu-1:, N. F. Osmotic Pressure, iiolecular Height, and (liability of Serum Albmnin. J. Biol Chem” pp, 555-77 aha-17k, N. F. Osmotic Pressure, Lioleculnr weight, and ‘12“: ability of Amandin and. Excelsin and Certain Other I‘oteins. J. Biol. Chem., 23.9.: 63-83 (1057). 3:411:44, N. F. Osmotic Pressure, Molecular ..-"eight, and Etability of Serum Globulin. J. Biol. Chem, 121, 975-402 (1937). (Burk, H. F. Osmotic Pressure, Ilolecular .“Ieight, and kStability of Gliadin. J. Biol. Chem., 124-, 49-70 ( Burk, N. F. Osmotic Pressure, molecular L‘Jeight, and Dissociation of Limulus Hemocyanin. J. Biol. Chem” 153, 511-520 (1940). Waugh, D. F. Protein-Protein Interactions. In "Advances in Protein Chemistry”, H. L. Anson, K. Bailey, J. T. Edsall, eds., Academic Press, Inc., New York, Ii. Y., vol. 9, 559-41.(1954). ‘ Bull, H. B. Osmotic Pressure of Egg Albumin Solutions. J. Biol. 011011., 157, 145-51 (1941). ‘ Schulz, G. V. Molecular .‘Ieight Determinations on 1101201093- ous Series of High Molecular .‘Ieight Polymers. Z. Physil—z. Cher-1., A 176, 517-57 (1956). ‘ .Bourdillon, J. An Apparatus for the Rapid. and Accurate Determination of Low Osmotic Pressures. J. Biol. Chem. 127, 617-625 (1959). - Hepp, O. A New Oncometer for Estimation of Colloid Osmotic Pressure with Increased Precision and Simplicity. Z. Ges. Exptl. Iled., g_9_, 709-17 (1955); CA, 51, 7078 ((757) “v :‘L’fl ‘3: l, w ,.-‘ -—————-—-——' 61. 62. 55. 54. 655 665 637 - €5£3~ 77 (lexzrter, s. 5., and Record, B. a. S3I? Solutions of Polysaccharide Derivatives, Part I, A 13:39? Form of Osmometer. J. Chem. Soc., 1939, 660-4 (1959). The Osmotic Pressure I“111035, R. M., and Head, D. J. Osmotic Pressure of Poly— Vinyl Chloride Solutions by a Dynamic Method. J. Phys. (311em., 41, 59-70 (1945). Ihundgren, H. P., and Ward, W. H. Molecular Size of i’roteins. In "Amino Acids and Proteins”, D. M. Green- TDerg, ed., Charles C. Thomas, Springfield, 111., V01. 1' 33p. 522-50 (1954). U—‘rl—q’l Efang, J. T., and Foster, J. F. Determination of Critical Ilicelle Concentration by Equilibrium Dialysis. J. Phys. Colloid Chem., 57, 628-55 (1955). Djang, S. S. T. "The Isolation, Fractionation, and L} Electrophoretic Characterization of the Globulins of ' 9EMng Bean". Unpublished Ph. D. Thesis, Michigan State College, pp. 75-80 (1951) Dunn, M. S. Casein. In "Biochemical Preparations", H. E. Carter, ed., John Wile and Sons, Inc., New York, N. Y., Alexander, A. E. and Johnson, P. "Colloid Science". _0x- _ ford University Press, London, Vol. I, pp. 171-5, 851,(1949L Wagner, R. H. Determination of Osmotic Pressure. Weissborger3"Physieal Methods of Organic Chemistry, Interscienee Publishers, New York, N.Y., Vol. 1, pp. 487-589 (1949). In Bull, H. B. and Currie, B. T. ‘Osmotic Pressure of Beta Laeto lobulin Solutions. J. Am. Chem. Soc., 68, 742-5, 1945 . Hawrowitz, F. "Chemistry and Biology of the Proteins". Academic Press, Inc. New York, N.Y., 6- (1950 0 Peterson, R. S. Effects of Disaggregation on the Separa- tion of Casein into its Components. Abstracts Papers Am. Chem. Soc., 128, 21 C (1957). Nitschman, H., and Lehmann, W. Electrophoretie Different- iation Between Acid Casein and Rennet Casein. Expor- '75. '74. 75. 76. 7'7- 78‘ 78 Clark, E. P. "Semimicro Quantitative Organic Analysisi', Academic Press, Inc., New York, N.Y. pp. 57-45 (1945). G‘Ornall, A. e. Barawill, c. J., and David, 91.1.1. Deter- mination of Serum Proteins b Means of the Biuret Reaction. 3 . Biol. Chem. 177, 751-66 1949). Hawk, P.B., Oser, B. L., and Summerson, W. H. "Practical Physiological Chemistry". The Blakiston Company, Inc., New York, N.Y., VP. 882-88 (1954). Bull, H. B. "Physical Biochemistry". John Wiley and Sons, Inc., New York, N.Y. pp. 523-4 (1951). Kipp, N. J., Groves, M. L., and McMcekin, T. L. Acid- Base Titration, Viscosity, and Density of Alpha, ‘Beta, ' and Gamma Casein. J. Am. Chem. Soc., ILA, 4822-6 (1952). Youden, W. J. "Statistical Methods for Chemists". John Wiley and Sons, Inc., New York, N.Y., pp 42-5 (1951). “‘.‘ “Tl APPENDICES APPENDIX I DATA OBTAINED FROM THE F"T"'OTIC 1". n ) J.,... '3 ES EUR E LEE :‘LSUR Eli-“713111" S O Thirty measurements were made which are arranged in c‘ . 1133‘ 0110105310 :11 order. The solvent is 6.66 I: urea buffered to 1311 4.8 with acct.th ionic strength 0.1 except in the o. q - 0 "Se of calcium-procipitated casein where he pl; 13 6.0. y ‘1 “crklilibrium osmotic pressure was determined from a plot of 0“. Q Served pressure v-‘rsus time. Protein concentration was die"terminal by absorbance at 280 mu before and after each n . 1% asuremont. 81 Osmotic Pressure Measurement No. 1 Acid-_P1:‘ecipitated Casein Po°c —_= 14.76 cm. H20 Tempo = 30° c. c :2 1.67m./100 ml. Bull osmometer used. Po/C = 8.84 Equib. P. = 16.58 cm. H20 Readings * Time Pressure Thee Pressure ’ (hr.) (cm. H20) (hr.) (cm. H20) 0 12.85 120 16.58 . 4 14.65 136 16.58 18 16.09 144 16.22 28 . 16.43 162 15.02 L: 42 16.54 187 15.10 g 51 16.27 . 195 15.72 68 16.99 197 16.00 76 16.90 212 15.60 87 16.75 216 ' 15.40 100 16.67 252 15.45 112 16.40 258 15.01 MM lof- 1 I n n 100 200 500 400 566 660 706 Hours 40- :30 343 O 1.1 Osmotic Pressure Measurement No. 2 Acid-“Precipitated Casein P060 .4 3.22 cm. H20 Temp; = 30° c. c = 0.74 gnu/100 ml. Bull osmcmeter used. Po/C = 4.36 Equib . 13.23.58 cm. HgO Results not used.':‘”“' Headings Time Pressure Time Pressure (hr.) (cm. 1120) (hm) (cm. H20) 1 7.43 171 2.57 10 7.53 191 2.88 24 5.75 197 2.86 31 4.98 210 2.76 44 4.24 216 2.71 51 4.02 245 2.60 70 3.67 257 2.56 76 3.58 280 2.52 94 5.45 288 2.54 101 5.44 311 2.39 121 3.34 341 2.44 142 5.17 554 2.11 166 '3.20& 377 2.09 167 1.73" 402 2.13 eHManometer reset "“Deviation of this point from regression is more than twice standard error of measurement. (5'? UL) Osmotic Pressure Measurement No. 3 Acid-‘Precipitated Casein Po°c.= 3.04 cm. H20 Temp- =-- 50° 0. c = 0. 57 gum/100 ml. Bull Osmometer used. P'-=O/C 8. Readings Time Pressure .1. Time Pressure (hm) (6:11.820) (hm) (mJIgO) 0 5.09 “— 166 5.59 10 5.44 178 3.23” 36 5.49 178 0.05" 39 5.21 181 0.06 43 5.01 204 0.12 59 4.51 233 0.15 68 4.38 256 0.15 85 4.01 283 0.11 91 3.92 301 0.10 111 3.33 > 322 0.07 134 3.38 335 0.11 155 3.37 *Manometer reset <¥<3 - 30 +- 19 E2c>r lo - NAI‘ 4 Air.“- ’ 1 l 100 200 300 400 500 500 766 Hours Ac id—Precipitated Cas sin 84 Osmotic Pressure Measurement No. 4 Po. 0; =11.88 cm. H 0 2 Temp- = 50° 6. c = 1.41 gnu/100 m1. Bull Omaneter.used.. Po/C = 8.42 Equib- P. = 13.18 cm. 320 _ Readings P"? Time Pressure Time . Pressure ' (hm) (cm. E1720) (hm) (cm. H20) 0 10.07 160 14.86 5 12.87 168 14.69 16 14.51 185 14.59 i 28 15.23 197 14,59 :4 41 15.70 215 15.94 52 15.85 254 15.57 66 15.75 259 15.18 76 15.78 284 12.57 91 15.63 507 10.48 110 15.58 551 10.26 159 14.98 $0 _ 25(3 — P 20 - 10 m l I l I l I 100 200 400 500 600 700 Hours 85 Osmotic Pressure Measurement No. 5 AC1d—Precipitated Casein Po°c. = 4-91 cm. H20 Telnp - = 300 C. c 20.59 gm./100 1211. Bull osmometer used. Po/C = 89 52 Equi‘b. P =5.45 cm. H20 Readings ; Time Pressure Time Pressure (hr.) (cm. H20) (hm) (cm. HBO) . L O 6.42 160 5.03 5 7.08 168 4.82 16 6.44 183 4.66 28 5.76 197 4.49 55 41 5.41 215 3.93 52 5.39 234 3.26 66 5.33 259 3.08 76 5.45 284 2.93 91 5.35 307 1.13 110 5.59 331 0.89 139 5.04 40F 30- I? 20— 10_. MN I l 100 200 500 400 500 WET—700 Hours Acid-Precipitated Casein Osmotic Pressure Measurement No. 6 Temp. = 30° C. Bull osmometer used. No squib. P. 40 50 2O 10 86 NO POD 0. value. 0 =0.29 gm./100 1111. No Po/C value. Readings Tune Pressure Time Pressure (hm (cm. 320) (hm) (cm. H20) 0 4.53 160 1.98 5 5.61 168 1.86 16 5.39 183 1.68 28 4.81 197 1.58 41 4.42 215 1.05 52 4.09 234 0.56 66 5.47 259 0.50 76 3.44 284 0.27 91 3.08 307 0.00 110 2.89 331 0.00 139 2.02 1 p- M A I I 100 200 300 400 500 600 700 .—,—..~ ‘- 2' H; 2. a. —v., . ##w b 87 Osmotic Pressure Measurement No. 7 Acid-Precipitated Casein Po°c. = 21.60 cm. I120 Temp. = 50° 0. c =---— 2.59 gnu/100 ml. Bull osmometer used. Po/C = 9.04 Equib. P.= 25.98 Readings Time Pressure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 0 17.21 185 23.90 21 22.35 209 23.98 41 25.47 209 15.67* 65 24.88 228 16.76 89 25.05 257 17.76 , 117 23.96 274 17.81 134 23.80 298 17.87 142 24.12 333 17.87 161 23.84 351 17.37 168 23.70 372 17.64 *Manometer reset 40F 50 ' P m 20 f I 10L 100 200 300 400 500 600 Hours 700 88 Osmotic Pressure Measurement No.8 Acid—Precipitated Casein Po°c = 12.32 cm. H20 Temp. = 50° c. c = 1.40 gm./100 m1. ilull osmometer used. Po/C == 8.80 Equib. P.== 13.68 cm. H20 Readings 7? Time Pressure Time Pressure ‘ (hm) (cm. H20) (hm) (cm. H20) 0 13.12 185 10.71 21 12.71 209 10.73" 41 ‘ 12.52 209 10.20“ E 65 12.99 228 4.05 S; 89 13.37 257 6.13 117 13.68 274 4.85 134 12.89 298 4.13 142 12.52 333 2.73 161 11.71 351 0.61 168 11.39 372 0.58 *Manometer reset 40 30 P 20 10 l I l I l l l 100 200 300 400 500 600 700 Hours 89 Osmotic Pressure Measurement No. 9 Acid-Precipitated Casein Pogo. =_- 4.93 cm. H20 0 Temp. =50° c. 0 =0.68 gn./100 ml. Bull osmometer used. Po/C =7.25 EQUib. P.==-5.47 Results not used.** Readings Time Pressure Time Pressure (bro) (Cm. H20) (hro) (cm. H20) 0 9.05 185 0.66 21 6.65 209 0.61” 41 5.76 209 6.94“ 65 5.47 228 5.07 89 5.06 257 0.92 117 5.15 274 0.39 134 4.11 298 0.37 142 3.53 333 0.39 161 2.52 351 0.33 168 1.62 372 0.33 40 30 2O 10 fyanometer reset ""Deviation of this point from regression is more than twice standard error of measurement. 100 200 300 400 500 600 700 Hours 90 Osmotic Pressure Measurement No. 10 Alpha Casein Po°c' -——-— 22.79 cm. H o o 2 Temp. 250° c. c =---- 5.00 gnu/100 m1. Bull osmometer used. Po/C = 7.60 Equib. 1:25.29 cm. H20 Readings Time Pressure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 17 19.01 190 25. 50 41 21.68 214 25.29 64 22.88 225 25.44 94 23.74y 242 25.58 118 28.65” 268 23.93 142 25.98 291 22. 56 166 25.72 *Constant temperature bath had overheated. “F 30.. P 20 - 10 L L .Li I I 1* I I 100 200 300 400 500 600 700 Hours 91 Osmotic Pressure Measurement No. 11 Alpha Casein P00 c. =14.89 cm. H20 Temp. =30° C. C = 2.00 gm/ZLOO m1. Bull osmometer used. Pb/C = 7.45 Equib. PZ=16.53 cm. H20 Readings T f .V Time Pre ssure Time Pre ssure 3‘ (hr.) (cm. H20) (hr.) (cm. H20) ‘ _, I 17 13.82 190 16.49 P 41 15.44 214 16.53 65 16.05 225 16.49 J 94 15.40% 242 16.40 g, 118 "18.43“ 268 14.72 142 17.02 291 15.41 166 16.87 *Constant temperature bath had overheated. 40F 10* 100 200 300 400 500 600 700 Hours 92 Osmotic Pressure Measurement No. 12 Alpha Casein No P000; value. Temp. =50° C. c = 1.00 gm./100 ml. Bull.0sm0meter used. No PO/C value. No equb.P, Readings Time Pressure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 17 8.76 190 5.51 41 9.44 214 4.88 65 9.69 225 4.66 94 9.74w 242 4.52 118 8.59“ 268 2.32 142 6.77 291 1.30 166 6.21 *Constant temperature bath had overheated. 40? 30- P 20- 10f 1L 1 I L 141 I I 100 200 500 400 500 600 700 Hours 93 Osmotic Pressure Measurement No. 13 Alpha Casein P e -==:12.82 cm. H20 0 c. Temp. = 30° C. C =‘-'— 1.68 gym/100 ml. Bull osmometer used. Po/C = 7.63 Equib. 2:14.25 cm. H20 Readings Time Pressure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 14 14.06 139 14.12 42 13.83 158 13.95 61 13.97 186 13.91 93 14.24 205 13.98 118 14.23 . 229 13.99 40[‘ 30 - P 20 ' W 10 ’ I I I L I I I 100 200 300 400 500 600 700 Hours 94 Osmotic Pressure Measurement No. 14 Alpha Casein Poo c. = 6.38 cm. 1120 Temp. = 30° C. C —-= 1.03 gym/100 ml. Bull osmometer used. Po/C =6.19 Equib. P. = 7.08 cm. 1120 Results not used.* Readings Time Pressure Time Pressure ’ 14 14.06 139 14.12 46 13.83 158 15.95 ’61 13.97 186 13.91 95 14.24 205 15.92 118 14.23 229 13.99 *Deviation of this point from regression is more than twice standard error of measurement. 40F 20’ 10' 100 200 300 400 500 600 Hours 1 A i’ 3 J - :- 95 Osmotic Pressure Measurement No. 15 Alpha Casein Po°c.=4‘60 cm. H20 Temp. =50° 0. c = 0.56 «gm/100 m1. Bull osmometer used. Po/C = 8.20 Equib. P. =5.lO cm. H20 Readings Time Pressure Time Pressure (hr. ) (cm. 1120) (hr. ) (cm. H20) 14 5.76 158 3.58 46 5.31 186 3.67 61 5.17 205 3.67 93 5.10 229 3.56 118 4.85 253 3.14 139 4.13 40 - 30 — P 20 ‘ 10 _ I I I I I l l 100 200 300 400 500 600 700 96 Osmotic Pressure Measurement No. 16 Beta Casein Poo 0.: 5.81 cm. 1120 Temp. =50° C. c ._— 1.54 {gm/100 ml. Bull oswemeter used. Po/C ==-4.33 Equib. P ===6.45 cm. H20 Results not used.* Readings Time Pressure Time Pressure {hr.) (cm. H20) (hr.) (cm. H20) 14 8.90 167 6.45 40 7.33 188 6.30 70 6.24 208 6.05 86 6.27 235 5.49 111 6.38 262 5.69 136 6.45 *Deviation of this point from regression line is more than twice standard error of measurement. 40 ' 30 ’ P 20 ' 10 pm I l I 1 I I !_.l 100 200 300 400 500 600 700 Hours 97 Osmotic Pressure Measurement No. 17 Beta Casein Po°c.= 9.32 cm. 1120 Temp. = 50° 0. c = 0.91 gm./100 m1. Bull osmometer used. Po/C =10. 25 Equib. P.= 10.35 cm. H20 E... Readings 7’ Time Pressure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 14 11.55 167 9.61 40 10.58 188 9.05 , 70 10.45 208 8. 57 pp 86 10.40 235 8.01 ' 111 10.35 262 7.37 136 10.13 40 " 30 P P 20 r 10 ’W I J l l I C I 100 200 300 400 500 600 700 Hours 98 Osmotic Pressure Measurement No. 18 Beta Casein Pooc;==-3;73 cm. H20 Temp. = 50° C. c = 0.55 gum/100 m1. Bull osmometer used. Po/C =10.66 Equib. P.= 4.14 cm. H20 Readings Time Pressure Time Pressure (hr. ) (cm. H20) (hr. ) (cm. 1120) 14 5.86 167 3.13 40 4.99 188 2.35 70 4.50 208 1.84 86 4.38 235 1.50 111 4.25 262 1.32 136 4.14 40 - 30 r P 20 7 10 P W | l I ll“ I 100 200 300 400 500 600 700 Hours Alpha Case in Temp. = 20° C. 40 10 99 Osmotic Pressure Measurement No. Pooc_=le.55 cm. H o 19 2 c = 2.51 {gm/100 m1. Bull osmometer used. PO/C = 8.07 Equib. P.=20.01 cm. H20 Readings Time Pressure Time Pressure (hm) (cm. HBO) (hr.) (cm. H20) 3 12.97 283 19.06 21 14.48 307 19.17 32 15.42 342 19.29 47 16.38 382 19.43 55 16.76 409 19.49 71 17.31 427 19.54 97 17.89 451 19.54 121 18.15 478 19.71 145 18.29 500 19.75 174 18.59 524 19.84 197 18.72 547 19.90 218 18.91 571 19.94 235 18.88 595 20.01 259 18.98 619 20.09 K. ."W: v—‘—: t 61—: ‘ e 3 t ; v; I I J I I I I 100 200 300 400 500 600 700 Hours :w— —.‘ m: J*-L_' “pm—m"?! ‘a vvrl ‘7‘. _—'—_,'.-'.'.V.'.' , 100 Osmotic Pressure Measurement N0. 20 Alpha Casein Po°c.= 9.86 cm. H20 Temp. 220° C. 0 = 1.75 gym/100 m1. Bull osmometer used. Po/C = 5.70 TEquib. P ===10.58 cm. H20 Results not usedfeé Readings Time Pressure Time Pressure (hro) (cm. H20) (111%) (cm; H20) 3 14.51 283 10.58 21 9.48 307 9.63 32 8.82 342 8.74 47 8.52 382 9.21 55 8.40 409 9.26 71 8.34 427 9.30 97 9.20 451 9.08 121 9.48 478 8.97 145 9.67 500 8.97 174 9.74 524 9.56 197 9.56 547 9.06“ 218 10.36 571 9.35" 235 10.26 595 8.72 259 10.29 619 8.60 *Manometer reset . . **Deviation of this point frdn regreSSlon is more 40r’ than twice standard error of measurement. 50 r— P 205 10W 4 1N»... I l I L I l ___I 100 200 300 400 500 600 700 Alpha Casein Temp. =20° C. Bull osmometer used. Equibl P. = 7.01 cm. HgO 40 30 20 10 .m!‘ “Reef-I; WI riw ‘ 2.2,.) 1.1.5.71 _ ,_ 101 _~~ 'W,‘ LX‘ '4» -1 ’T—"-‘-, Osnotic Pressure Measurement No. 21 P000; =6.53 cm. H20 C = 0.77 gem/100 ml. P.,/C =s.49 I I 300 400 Hours I 500 Readings Time Pressure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 3 8.58 283 6.82 21 9.03 307 6.80 32 8.50 342 6.82 47 7.92 382 6.82 55 7.70 409 6.84 71 7.43 427 6.84 97 7.18 451 6.87 121 5.95 78 6988 145 6.85 500 6.90 174 6.92 524 6.92 197 6.93 547 6.95 218 7.01 571 6.98 235 6.99 595 6.99 259 6.88 619 7.00 P L 600 _J 700 Alpha Casein 102 Osmotic Pressure Measurement No. 22 Poo 0. := 15.40 cm. H20 Temp. = 20° C. C = 1.98 Emu/100 m1. Fuoss-Mead osmometer used. PO/C = 7.78 Equib. P.==-16.53 cm. H20 Readings Time Pressure Time Pressure (h-ro) (cm. H20) (hrs) (cm. H20) 0 17.94 255 16.76 ' 21 14.12 259 17.28 32 15.73 283 16.98 47 13. 89 307 17. 31 50 14.17 342 17.27 55 14.65 382 17.11 65 16.20 409 7.11 71 16.54 427 17.20 89 17.70 478 16.39 97 17.88 500 16.60 121 16.70 524 16.49 137 16.53 547 16.38 145 16.51 571 16.43 174 16.62 595 14.69 197 16.69 619 14.82 218 16.82 40 " 50 F P 20 ~ 10 L- I I l L 1 l #1 100 200 300 400 500 600 700 Hours Alpha Casein Temp. =10° C. 40 10 103 Osmotic Pressure L'ieasurement Ho. 23 Pooc‘. 228.64 cm. H Hours 0 = 5.80 gm./100 ml. Fuoss-Iéead osmometer used. Po/C 7.54 Equib. P: 29.69 cm. 1120 Readings Time Pressure Time Pressure (hrs) (cm. H20) (hrs) (cm. H20) 0 28.75 92 29.49 ' 11 26.35 108 29.89 14 27.01 116 28.84 16 27.05 132 29.48 19 27.24 155 29.66 24 27.61 140 29.04 35 28.53 155 29.38 43 29.00 156 29.69 49 29.16 160 29.40 61 29.76 180 30.20 73 29.42 183 29.58 84 29.39 50 K W’ - ' 200 400 500 Alpha Casein Temp.==10° C. Fuoss-Jead osmcmeter'used. Equib. P ==2 .31 cm.H20 104 Osmotic Pressure Measurement No. 24 Poo c. = 20.56 cm. H20 3 Po/C ll 8.49 Results not used."" 40— 30+ 10L sor :fifieset manometer . . ““Deviation of this noint from P82383810n is more Readings Time Pressure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 0 13.14 104 21.31” 5 14.24 119 17.44“ 11 14.95 125 19.73 27 17.64 132 19.45 56 18.62 148 19.19 47 19.58 152 19.04 56 18.74 176 18.88 75 21.73 194 18.20 81 21.52 216 17.04 96 21.57 than twice standard error of measurement. 300 Hours 1 400 500 600 2.42 gnu/100 m1. 700 Osmotic Pressure Measurement No. Beta Casein Temp. =10° C. 105 190°C. =55.90 cm. 1120 C 25 3.00 {gm/100 ml. Fuoss-Iviead osmometer used. Po/C =1l.97 Equib. P 237.22 cm. 1120 Readings Time Pressure Time Pressure (hro) (cm. H20) (bro) (cm. H20) 0 19.06 71 36.09 i 4 20.88 88 36.35 16 27.49 100 36.91 20 29.24 114 67.12 24 30.08 121 37.24 40 31.04 138 37.46 44 51.95 144 57.12 48 32.52 163 37.11 52 33.40 175 37.25 64 35.31 1C .J.___.....--.L..._.._.__.-__.L1 i 4 l 100 200 300 400 500 600 Hours Osmotic Pressure Measurement No. 26 Beta Casein 13000.: 6.22 cm. H20 Temp. ==10° c. c =2 0.61 gm./1OO m1. Fuoss-Mead osmometer used. Po/C ==lO.2O Equib. P -= 6.45 cm. H20 Readings Time Pressure Time Pressure (hr.) (Cm. H20) (hr.) (cm. H20) 0 5.19 166 6.62 8 81 5.32 193 5.50 99 5.71 200 6.13 109 5.93 219 6.45 120 6.16 247 5.81 128 6.14 277 6.13 144 6.57 289 6.43 156 5.64 40- 30“ P 20’ 10" 1 1 1 L 4 L 1 100 200 300 400 500 600 700 Hours Alpha Casein Temp. = 20° C. 40 113 99 Pooc_=1s.66 cm. H o Osmotic Pressure Measurement No. an”. 7‘3..- 2. '1 19 2 c = 2.L1 {gm/100 m1. Bull osmometer used. Po/C = 8.07 Equib. P.=20.01 cm. H20 Readings Time Pressure Time Pressure (hr.) (cm. HBO) (hr.) (cm. 1120) 3 12.97 283 19.06 21 14.48 307 19.17 32 15.42 342 19.29 47 16.38 382 19.43 55 16.76 409 19.49 71 17.31 427 19.54 97 17.89 451 19.54 121 18.15 478 19.71 145 18.29 500 19.75 174 18.59 524 19.84 197 18.72 547 19.90 218 18.91 571 19.94 235 18.88 595 20.01 259 18.98 619 20.09 I 1 J L 1 l 1 100 200 300 400 500 600 700 Hours 100 Osmotic Pressure Measurement No. 20 Alpha Casein Po°c.: 9.86 cm. H20 Temp. =20° C. C = 1.75 gnu/100 ml. Bull osmometer used. Po/C = 5.70 Equib. P ===10.58 cm. H20 Results not usedias Readings Time Pressure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 5 14.51 283 10.58 21 9.48 307 9.63 32 8.82 342 8.74 47 53.52 322 9.21 55 8.40 409 9.26 71 8.54 427 9.30 97 9.20 451 9.08 121 9.48 478 8.97 145 9.67 500 8.97 174 9.74 524 9.56 197 9.56 547 9.06% 218 10.36 571 9.35" 255 10.26 595 8.72 259 10. 29 619 2. 5° *Manometer reset . ,0 **Deviation of this point frmn regr9351on 19 more 40r than twice standard error of measurement. so, P 20r 10 W 3 : : ;~N"‘H l l 1 1_ l j .750 100 200 300 400 500 600 Alpha Casein Temp. =20° C. Bull osmometer used. Equibl P. = 7.01 cm. HOD 101 Osmotic Pressure Measurement No. 21 P000; =G.53 cm. H20 C P.,/0 =e.42 == 0.77 gm./100 m1. Readings Time Pressure Time Pressure— (hr.) (cm. H20) (hr.) (cm. H20) 5 8.58 283 6.829.— 21 9.05 507 6.80 52 8.50 342 6.82 47 7.92 582 6.82 55 7.70 409 6.84 71 7.45 427 6.84 97 7.18 451 6.87 121 5.93 472 6.88 145 6.85 500 5.90 174 6.92 524 5.92 197 6.93 547 6.95 218 7.01 571 6.98 235 6.99 595 6.99 259 6.88 619 7.00 40[* 30'- P 20" 101- -511 N4 4 1 _ -# . - ;4_ : - :r ‘ t ' ' L4 ' ' J 1 600 753 100 200 500 400 500 Hours Alpha Casein 102 Osmotic Pressure Measurement No. 22 P00 0. 215.40 cm. H20 ‘ _. o - . Temp. - 20 C. c = 1.98 gnu/100 m1. Fuoss-Mead osmometer used. Po/C = 7.78 Equib. P ===16.53 cm. H20 Readings Time ‘fiwPreesure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 0 17.94 255 16.76 9 21 14.12 259 17.28 52 15.73 283 16.98 47 13.89 307 17.31 50 14.17 342 17.27 55 14.65 382 17.11 55 16. 20 409 17.11 71 16.54 427 7.2 89 17.70 478 16.39 121 16.70 524 16.49 137 16.53 547 16.58 145 16.51 571 16-43 174 16.62 595 14.69 197 16.69 619 14.82 218 16.82 40(- f 30'- P 20 10 100 200 300 400 Hours 500 600 700 Alpha Casein Osmotic Temp. =10° C. Fuoss-Mead osmometer 103 Pressure Measurement No. 23 used. Equib. P2.- 29.69 cm. P120 40 10 Pooc; 228.64 cm. H C Po/C = 7.54 2 0 '== 3.80 gm./100:m1. Readings Time Pressure Time Pressure 0 28.73 92 29.49 9 11 26.35 108 29.2 14 27.01 116 28.84 16 27.05 132 29.48 19 27.24 135 29.66 24 27.61 140 29.04 35 28.53 155 29.38 43 29.00 156 29.69 49 29.16 160 29.40 61 29.76 180 30.20 73 29.42 183 29.58 84 29.39 F 1 J I I AJ 400 500 600 700 200 300 Hours 104 Osmotic Pressure Measurement No. 24 Alpha Casein Pd°c 220.56 cm. H20 Temp.==10° C. c. --= 2.42 gym/100 ml. Fuoss-Jead osmometer*used. Po/C :== 8.49 Equib. P ==91.31 CmQHQO Results not'used.** Readings Time Pressure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 0 15.14 104 21.51.__ 5 14.24 119 17.44" 11 14.95 125 19.75 27 17.64 152 19.45 56 18.62 148 19.19 47 19.58 152 19.04 56 18.74 176 18.88 73 21.73 194 18.20 81 21.52 216 17.04 96 21.57 fiReset manometer 0 **Deviation of this point from reoression.is more than tvlice standard. error of measurement. 40— 31 10- ‘ ‘“J ‘4 1‘ i 506 700 100 200 300 400 500 Hours 1C 105 Osmotic Pressure Measurement No. 2 5 Beta Casein Po°c. =55.90 cm. 1120 Temp. =10° C. 0 = 5.00 gem/100 m1. F‘uoss-Iviead osmometer used. Po/C =11.97 Equib. P 237.22 cm. 1120 Readings Time Pressure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 0 19.06 71 56.09 ' 4 20.88 88 36.35 16 27.49 100 36.91 20 29.24 114 37.12 24 30.08 121 37.24 40 31.04 138 37.46 44 31.95 144 37.12 48 32.62 163 37.11 52 33.40 175 37.25 64 35.31 J— l --..L 1 l I 100 200 300 400 500 600 700 Hours “.f Beta caseinl ‘ - 0 Teztp, -10 floss-Mead Equib,‘ p : Time (hr.) i 81 99 109 120 1138 144 155 40 ‘ "» 5"" —:‘" .‘R‘ w" t-vn-‘a—z"“=sl_=rx- 1’ —. i 731““- q‘ " _.-1_T < A "J, " —~.1\,: Tara 29.4 . .~. -.. ' “g.- =o~ * *4?; '~ . a ‘ 7 - Osmotic Pressure Measurement No. 26 Beta Casein Po°c.== 6.22 cm. H20 Temp. =10° c. c = 0.61 gnu/100 ml. Fuoss—Mead osmometer used. Po/C ==10.20 Equib. P = 6.45 cm. H20 Readings Time Pressure Time Pressure (hr.) (cm. 1120) (hr.) (cm. H20) 0 5.19 168 6.62 81 5.32 193 5.50 99 5.71 200 6.13 109 5.95 219 6.45 120 6.16 247 5.81 128 6.14 277 6.15 144 6.57 289 6.45 156 5.64 40— 30* P 20” 10' W l | 1 L 4 l J 100 200 500 400 500 600 700 107 Osmotic Pressure Measurement No. 27 Beta Casein Po°c. =20.32 cm. 1120 Temp. = 10° 0. c = 1.84 gun/100 m1. Fuoss-Mead osmometer used. Po/C -==11.05 Equib. P = 21.07 cm. H20 Readings Time Pressure Time Pressure (hr.) (cm. H20) (hr.) (cm. H20) 0 12.68 121 21.08 12 12.17 146 20.26 24 17.00 152 20.64 32 17.87 157 21.02 38 18.72 170 21.20 53 19.81 181 20.92 77 20.70 191 21.07 99 20.72 276 20.88 109 20.73 288 21.38 40 r- 30 L P g l _ l ,L L I I 100 200 300 400 500 600 700 Hours 108 Osmotic Pressure Measurement No. 28 Calcium-Precipitated Casein P 0 ~ 215.85 cm. H20 o 0. Temp. =10° c. c = 1.77 gun/100 m1. Fuoss-Head osmometer used. Po/C == 8.95 Equib. P ==16.43 cm. H20 Readings Time Pressure Time Pressure (hr.) (cm. 1120) (hr.) (cm. H20) 0 18.38 55 16.25 5 17.59 72 16.43 10 16.66 152 15.89 11 17.01 167 16.04 20 16.38. 174 15.50 29 16.42 287 15.71 45 16.05 4,0 _, 30 ' P 10 - 1 1 1 1, 1 I 100 a 200 300 400 500 600 700 Hours Calcium-Pr Temp. = 10° Fuoss-hie ad Equib. P = 30- 20- 10 109 Osmotic Pressure Measurement No. 29 Calcium-Precipitated Casein Po°c.‘==5970 cm. H20 Temp.==10° c. 0 == 0.70 gm./100 m1. Fuoss-Mead osmometer used. Po/C == 8.14 Equib. P ==5.91 cm. 320 Readings Time Pressure Time Pressure £315) (cm. HZO) (hr.) (cm. H20) 0 8.74 85 5.91 16 6.23 93 5.35 25 6.04 111 6.03 57 5.65 118 5.17 45 5.33 121 5.42 62 5.72 135 5.75 73 5.84 138 5.54 40* 50!- P 20r- 10f \ l 1 1 _L l 1 100 200 300 400 500 600 700 Hours Calcium—1 Temp. = 1‘ E‘uoss—Iie a Equib. P = 20'- “, 71:) '17.; w ‘- .: 4 ~- «- 110 Omnotic Pressure Measurement No. 30 Calcium-Precipitated Casein Pd’c. Temp. ==10° c. .— ~ = 32.53 cm. 1120 5.54 gm./100 m1. Fuoss—Mead osmometer used. PO/C == 9.74 Equib. P:= 33.72 cm. H20 Readings Time Pressure Time Pressure (hr. ) (cm. H20) (hr.) (cm. H20) 0 34.00 148 33.86 7 33.98 168 33.82 23 34.11 173 34.05 34 34.44 192 33.80 75 34.17 202 33.86 96 36.92 214 34.30 121 33.72 220 33.74 142 34.62 242 33.95 40!- 30* 2d- 10r 1 it I 1 L L 1 100 200 300 400 500 600 700 {N APPENDIX 11 AN EXAKPLE OF THE STATISTICS USED IN DETERMLNING OSICTIC BEHAVIOR AND MOLECULAR UEIGHT The statistical treamuents used are discussed by Youden (78). Using acid-precipitated casein as an example, the follow- ing values are obtained from the experimental data: c Po/C 02 Po - . n == 6 0.57 8.23 0.14 5.04 20 = 7.83 0.59 8.32 0.55 4.91 U = 1.30 1.40 8.80 1.96 12.50 (26 ‘3 261.31 1.41 8.42 1.99 11.88 Ere/c 51.65 1.67 8.84 2.79 14.76 m 8.61 2.59 9.04 5.71 21.60 202 12.94 ZEPO 68.49 The slope of the least squares regression line for the Po/C versus C plot of this data is given by the formula: b .-. nzpo - going/C) nicr-(ECW 6 x 68.49 - 7.83 x51.65 6 x 12.94 -— 61.51 = 0.599 . The Po/C intercept (a) of the least squares regression line :18 given by: a 2 (P070 -b?;' = 8.61 - .599 x 1.50 = 8.08 the 901111.131 in which 4 1.0m the 5A 2 Value andS Thus, the least squares regression for this data is, Po/C = 8.084—0.5990 The standard error of est rate is the root mean square of the PO/C deviations about a fitted curve and is defined by the equation: 3%: 242 n in whichMA is the deviation of a given experimental point form the least squares regression line. Calculating the 2 A 2 value, Po/C Po/C 4s zf' 8.23 8.25 0.00 0.0000 8.52 8.32 0.00 0.0000 8.80 8.64 0.16 0.0256 8.40 8.64 0.24 0.0576 8.84 8.75 0.09 1 0.0081 9.04 9.05 0.01 0.0001 2A2 : 0.0914 3 - .0914 = .125 2 ' "7?“ a value of .123 is obtained for the standard error of measure- and substituting, ment. 7 ’l k“ “x K might 'v' “.1118 - a Q 111118, tile Pr . L15 (1.th L B? 3111:, 139-8635’ a? acm'PI‘ec i... 113 The standard deviation of the intercept (:3) was the basis for the plus or minus of the determined molecular weight value. It is given by: 2 _ i Ba?" 8" nZC’*— (265'; :— .1.'33 12.94 a Y‘Irlogz -‘ 61:001— = .110 Thus, the Po/C intercept of the least squares regression for this data is 8.08 with a standard deviation of 0.11. By substituting 8.08 into the van't Hoff equation on pageab, a molecular weight value of 28,700 is obtained for acid-precipitated casein. The upper and lower limits of 1400 for this value are obtained by substituting 7.97 (8.08 — 0.11) and 8.19 (8.08 +0.11) into the van't Hoff equation. ”:31. em”, , » ‘TL. .‘uw; , i I 5"! MICHIGAN 9”“, WWQSH'Y ob #61681de 4.. AEHCE DEPARTMENT OF CHLMiSTRY EAST LANSING, MICHIGAN "‘441414441“