HENRYB. BULLAND BYRONT. CURRIE
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Vol. 68
propenyl grouping is accompanied by a shift of bestrol and of 3,3'-propcnylhexrstrol arc a t identithe absorption maxima in the ultraviolet. The cal wave lengths. absorption maxima of 3,3'-propenyldiethylstil- CHICAGO,ILLINOIS RECEIVED J A N U A R Y 5, 1946
[CONTRIBUTION FROM
THE
DEPARTMENT OF CHEMISTRY, NORTHWESTERN UNIVERSI~ Y MEDICAL SCHOOL]
Osmotic Pressure of @-LactoglobulinSolutions BY HENRYB. BULLAND BYRONT. CURRIE' through t h e courtesy of Dr. C . J. 13. Thor. The moist casing was securely knotted at one end and the other end was slipped over a rubber stopper which had bcen trimmed to size, and attached t o the end of the tube which was to contain t h e protein. T h e stopper had been previously coated with stopcock grease. The end of the sack which had been slipped over t h e rubber stoppcr was wrapped TABLE I tightly with a rubber band t o complete the attachment t o MOLECULAR WEIGHTOF LACTOGLOBULIN AS REPORTED the rubber stopper. T h e sack was thcn filled with pro:ciii solution through the larger glass tube t o which !vas a t BY VARIOUSWORKERS tached a second, larger rubber stopper which was coated Molecular with stopcock grease. The apparatus was then asMethod weight Workers sembled as shown in Fig. 1. T h e larger rubber stopper Ultracentrifuge (equilibrium) 38,000 Pedersen" was held in place by a metal clamp which is n o t shown in and diffusion Fig. 1. Small one-hole rubber stoppers were placed in the Ultracentrifuge (rate seditubes containiiig the outside solution and the protein solumentation) 41,500 Pedersen" tion. Their purpose was to decrease the evaporation of );-Ray (wet crystals) 33,000 Crowfootb these solutions during a n experiment. McMeekin and Wiariier' The wholc apparatus was clamped in a fixed position X - R a y (air dried crystals) 35,000 Crowfootb in a constant temperature bath a t 25". The stopcock McMeekin and Warner' was allowed t o remain open until the apparatus had come Chemical analysis mini42,000 Brand and Kasseld t o temperature. The position of the toluene level in the Chemical analysis mum 42,000 Chibnall' capillary was marked by appropriate means and the stop 42,020 Brand el d' Chemical analysis (m. w , ) cock was closed. The toluene meniscus immediately be38,000 Gutfreundv Osmotic pressure gan t o rise or fall depending on whether the hydrostatic 2 X 17,100 Bullh Film pressure previire was greater or less than the osmotic pressure. 35,000 Iieller and Klevensi Light scattering Protei:i solution was added t o or removed from the protei11 Osmotic pressure 35.020 Bull and Currie solution column t o bring the toluene meniscus t o its original Crowfoot, level. Adjustinent of the protein solution level was made O1 Pedersen, Biochem. J . , 30, 961 (1936). Chew. Rev., 28, 215 (1941). McMeekin and Warner, from tiitie t o time as needed. The toluene column thus THISJOURXAL,64, 2393 (1942). Brand and Kassel, acted essentially as an indicator and in the ideal case there J . Biol. Chem., 145, 365 (1942). ' Chibnall, Proc. Rny. is no net motion of liquid across the sausage casing memSoc. Londm, B131, 136 (1942). f Brand, Saidel, Gold- hrane. Usually, no further adjustment of the protein water, Kassell and Ryan, THISJOURNAL, 67, 1.524 (1945). solution column was necessary after about two hours. I n Bull, THISJOURGutfreund, Nulure, 155, 237 (1945). all cases, however, t h e experiment was allowed to continue NAL, 68,.745 (1946); Heller and Klevens, private com- overnight. A t the end of this tiinc, the positioii of ~ l i c munication. toluene column had usually shifted one or two nim. The drift of the toluene level was mi!!tiq"cJ by the density of The present paper reports the result of 20 toluene and applied as a correctio:: t o the osmotic pressure. difference in lcvcl between the outside solution and osmotic pressure measurements on solutions of P- The the protcin solution \vas measured with a catlictoinetcr. lactoglobulin along with the calculation of the This difference iii lcvcl whcn niultiplicd by the density of molecular weight of this protein. the protein solution gave, after applying the correction d u c to the small excursion of the tolu('!ie colunln, the osmotic pressure of thc protein solutiori 1 1 1 i ! .'iiicters of water. Experimental The P-lactogloliulin Mas prepared by a modification of a T h e osmotic pressure apparatus was a modification of method suggested in a private coiiiniunication by Dr. A. t h a t described by Bull.* This modification is diagrammed H. Palmer. Raw whole niilb was b r o ~ l g i ~t ot 60% saturain Fig. 1. T h e capillary tube had an inner diameter of 0.2 mm. and was furnished by Corning Glass Works. tion with arnmoniuni su1Iatc. Thc solution was filtered About 5 CC.of the outside solutipn (solution whose com- and the filtrate brought t o 807, saturation with aiiiiiioposition was identical with t h a t of the protein solution niuin sulfate. A siiia11 ainouiit of water a l o i ~ g\\itli some toluene was aticlctl t o thc precipitate. 'The precipitate except for the absence of proteiii) was placed in the botwas transferrctl t o .;aus:ipe caGiiig w i r l c i i u l y z d agaiiist Iretom of t h e apparatus. Toluene was added from the top of the clean, dry capillary and forced dowii the capillary quent changes of rlistillcd \rat('r a t ;7' for four or five days. with gentle air pressure. Suction was then applied t o the T h e protcin so1utio:r was th-ii rrniovrd Iroiii the sausage capillary t o remove the trapped air. T h e apparatus was casing and any cstr:riieous snlid iiiatcrial filtered off. The then filled with outside solution. T h c sack which coii- filtrate was adjustcid to PH 5.1 by the cautious addition of tained t h e protein solution was made from Visking sausage dilutc hydrochloric acid. The turbid so!ution was then casing whose flat width was 1.5 cin. and was supplied dialyzed agaiiirt distilicu water and the dialysis contiiiued until 110 furthvr scl>aratiwi of the "oily" phasr tool; place. ( 1 ) On leave ol absence from the Corn Products llefinitiy Coni T h r protcin was thew crystallizvrl as ticscribeti by Pa1111cr.~
A number of values for the molecular weight of P-lactoglobulin from cow's milk have been reported. There is, however, a disconcerting lack of agreement between these values as is shown in Table I.
1
*
.
paw. (2) Bull. J . B i d Ckcm.. 137, 1.13 (1'311)
( 3 ) l ' , ~ I i i i c r ,i b i d . . 104, 3 3 3 ( l ' J 3 4 j
May, 1946
OSMOTICPRESSUREOF @-LACTOGLOBULIN SOLUTIONS
As will be pointed out later, the recrystallization of Blactoglobulin by the addition of dilute sodium hydroxide t o produce a pHi of 5.8 followed by subsequent neutralization with dilute hydrochloric acid does not, in our hands, yield a product satisfactory for osmotic pressure measurements. After recrystallizing twice according t o Palmer's method, the following additional crystallization was made. T h e crystals of @-lactoglobulin were dissolved in 0.07 M sodium chloride. The turbid solution was centrifuged and the clear solution dialyzed against distilled water with frequent agitation so that many small plate-like crystals were produced. This type of recrystallization was repeated several times. The final crystals, when dissolved in sodium chloride .solutions, gave, water-clear solutions up to a protein concentration of 6%. For the osmotic pressure measurements, the 8-lactoglobulin solutions were made up in such a way t h a t the final coiicentratlon of sodium chloride was 0.5 M . T h e concentrations of @-lactoglobulinsolutions were determined with a dipping refractometer (Zeiss). The refractive increment was measured on eleven different protein solutions whose concentration extended from 0.745 g. per 100 cc. t o 6.175 g. per 100 cc. of solution. The concentrations of these solutions were obtained by drying the solutions t o constant weight ill a vacuum oven a t 105" and subtracting from these weights the known weight of sodium chloride present. T h e concentration of protein in grams per 100 cc. of solution was then calculated by the empirically four,d relation C = 0.2121A - 0.00024Aa where A is the dipping refractive increment of the protein solution above that of 0.5 21.1 sodium chloride. Fresh solutions from the protein crystals were prepared for each osmotic pressure measurement. Two separate lots of /3-lactoglobuliii were used; the data from these two lots were iiidistinguiishable. Osmotic pressure measurements were also made on successive recrystallizations. T h e p H of the protein solutions were betrveen 5.1 and 5.2 and the protein acted as its own buffer t o maintain the PH constant. The osmotic pressure of 8-lactoglobulin was also measured in the presence of a series of concentrations of urea. These solutions were prepared from a mother solution of @-lactoglobuliIi and m a i ~ t a i n e da t room temperature for x v e r a l days prior to the osmotic pressure determination. The sodium chloride concentration in these solutions was 0.5 M.
743
h' Protein Solution
1;1 6
Toluene
Fig. l.--Osrnotic pressure apparatus.
to decrease. It was concluded that the sodium hydroxide-hydrochloric acid technique tends t o produce a heterogeneous product by virtue of local accumulation of alkali as the sodium hydroxide is added to the protein. All data obtained Results on &lactoglobulin recrystallized by the sodium Twenty-three osmotic pressure measurements hydroxide-hydrochloric acid technique were rewere made on 0-lactoglobulin recrystallized by jected as unacceptable. adding dilute sodium hydroxide to produce a pH Table I1 gives the osmotic pressure and the calof 5.8 and thcn neutralizing the alkali with dilute culated molecular weight of P-lactoglobulin, hydrochloric acid followed by subsequent di- These measurements were made on water clear alysis. These nieasurements were very unsatis- solutions of the protein which were prepared from factory as there was a wide spread of values and protein recrystallized by dialyzing sodium chloosmotic equilibrium appeared to be approached ride solutions of the protein against distilled very slowly. Indeed it is doubtful in a number of water. Table I1 includes all data obtained on cases that equilibrium was ever attained. It was these solutions and no results have been renoted that solutions of the protein re~rystallized jected. The osmotic pressures of most of the by the sodiurn hydroxide-hydrochloric acid pro- solutions were measured in duplicate. The cedure were nevcr entirely clear. Repeated re- concentrazion of protein is given in grams of crystallizations by this technique failed to im- protein per 100 grams of 0.5 M sodium chloride prove the situation. Tlie average of the twenty- solution. three detcrminations gave a molecular weight of The average molecular weight is 35,050 with a 40,500, with si standard deviation of the mean of standard deviation of the mean of 144. 2,000. It appears from the ultracentrifugal The results for the osmotic prcssure of 0studies of f'edersen4 that P-lactoglobulin is lactoglobulin in the presence of urea are given peculiarly sensitive to alkali. Bveri at n pM as in Table 111. low as 7.5 the sediriiciitatioii constarit had begun Solutiotis containing 7 and S ilf urea were dis(41 l i d c r c i i c e of T u b k 1 tinctly turbid,
HENRYB.BULLAND BYRONT.(3mt.1~
744
Vol. 68
The reason for this disagreement is to some extent obscure. Our work indicates that @-lactoglobulin has considerable tendency to undergo 25 reactions which Iead to a heterogeneous product. Protein Osmotic rea.. cm. of Mol. wt. conm. The recent paper by Briggs and Hull' sub34,860 1.485 10.95 stantiates this conclusion. It is our belief that 11.03 34,600 1.485 much of the difficulty associated with an accurate 34,600 1.931 14.35 determination of the molecular weight of @14.28 34,760 1.931 lactoglobulin can be explained on this basis. 12.31 33,890 1.622 It had been reported" that the spread film 34,530 2.328 17.33 molecular weight was about 44,000. This value 10.46 34,860 1.418 was obtained on a sample of protein which had 10.53 34,630 1.418 been recrystallized by the sodium hydroxide17.35 34,550 2.331 hydrochloric acid technique and was probably 17.30 34,630 2.331 aggregated. The spread film molecular weight of 35,380 I.346 9.78 8-lactoglobulin has been reinvestigated and is the 9.91 34,900 1.346 subject of a separate paper.9 As noted in Table 12.79 35,120 1.747 I the molecular weight of spread films of this pro35,350 1,747 12.71 tein prepared according to the present procedure 36,790 1.848 12.91 is 17,100 which indicates that p-lactoglobulin dis13.40 35,450 1.848 sociates into 2 fragments on the surface and twice 35,990 1.281 9.15 this weight is 34,200 in good agreement with the 9.32 35,320 1.281 osmotic pressure studies. In the presence of 14.42 35,170 1.972 Cu++ ions @-lactoglobulindoes not dissociate and 14.26 35,560 1.972 its film molecular weight is 34,300. Heller and Klevens'O measured the molecular TABLEI11 weight of @-lactoglobulin by the light-scatter OSMOTIC PRESSURE OF @-LACTOGLOBULIN IN THE PRESENCE method using a sample of protein which we supOF UREA plied. They state that their value is probably Protein accurate to plus or minus 1,000. concn., Molar urea g. per 100 g. Osmotic press., It can be seen from Table I1 that there is a concn. of solvent cm. of &O Mol. wt. tendency for the molecular weights in the first part 0 ... .. 35,000 of the table to be lower than those in the last part 2 0.977 8.46 30,500 of the table. The sequence of the values in the 3 .965 9.04 28,600 table is the sequence in which the osmotic pressure 4 .953 9.06 28,500 determinations were made. The actual work ex5 .468 5.06 25,400 tended over a period of about a month which 6 .464 5.95 21,700 means that the last determinations were made on 7 .457 4.93 26,100 solutions which were prepared from aged crystals. > 30,000 8 .452
