July 5, 1960
DISTRIBUTION STUDIES WITH HUMAN PLASMA ALBUMIN
group was noted when the reduction was carried out with a 50-mole excess of thioglycolic acid but with a 100-mole excess the treated albumin had 1.2 mole of sulfhydryl group. With a 200-mole excess, the treated albumin had 3.0 mole of sulfhydryl group and also contained 16% polymer. The treated albumins were converted to mercurial derivatives. Figure 3 was obtained with albumin treated with 100-mole excess of thioglycolic acid, and Fig. 4 was obtained with albumin treated with 200-mole excess of thioglycolic acid. The band with a K of 1 present in the untreated sample had disappeared, but a new band had appeared with a K of 1.6 to 2.0. Material isolated from the region with a K of 1.6 to 2.0 was estimated to be composed of 70% monomer, 14% dimer and 16% polymer. The albumin recovered from the main bands in Fig. 3 and 4 was combined and redistributed to give the patterns in Fig. 5. Here the small band with a K of 1 reappeared because of the tendency of the mercury derivative to dissociate as discussed above. When the albumin was treated with thioglycolate a t pH 9.0, i t showed 4.7 mole of sulfhydryl group The distribution of the mercurial derivative of this sample gave the pattern shown in Fig. 6. Based on the albumin band ( K = 1.3) in the distribution pattern, i t was calculated that about 15% of the albumin in the reduction mixture was the unaltered albumin but 50% had formed insoluble polymers. The reduction of the disulfide bonds of the albumin could ccnceivably occur by two distinct pathways, (a) the susceptible disulfide linkages of all the albumin molecules cleaved one a t a time with essentially the same rates or (b) the initial cleavage of one disulfide linkage of an albumin which could then lead to the explosive cleavage of the remaining susceptible linkages present in the same molecule. In the above reduction experiments, the albumin concentration was held a t a constant value but the thioglycolate concentrations and PH of the solutions were varied. In all cases, significant amounts of apparently unchanged albumin could be recovered from the reaction [CONTRIBUTION FROM THE
3355
mixture. While a detailed analysis of the products was not possible due to formation of polymers, the results suggest that reaction pathway (b) is favored and that cleavage of the first linkage of the albumin introduced new labilities to the protein structure. Another possible reaction with the thioglycolate treatment would be in the formation and equilibration of albumin isomer(s) due to the migration of the sulfhydryl group in the chain. This is suggested by the sulfhydryl group value of one mole per mole of albumin isolated from thioglycolate treated sample in Fig. 5, as compared with the value of approximately 0.9 from untreated albumins in Figs. 1 and 2. Such reaction products would not have been differentiated if the new isomers had K values similar to that of the original albumin. In the case of human plasma albumin as described in the following paper,22there seemed to be definite evidence for this type of interchange. The reduction experiments support the belief that the band with a K of 1 and the shoulder on the right edge of the main band were products of sulfhydryl-disulfide reactions of the albumin, thus indicating that the observed broadness of the albumin bands did not result from some slow disorganization of the secondary structure by the organic solvents. This observation was fully substantiated by the measurements of the rotatory dispersion and sedimentation rate of the recovered albumin which indicates that i t had retained its native secondary structure, shape and molecular size. In terms of countercurrent distribution, those observations implied the possibility of fractionation of many proteins in these near-critical point, high-salt content solvent systems if their reactive functional sites could be blocked. It perhaps should be stressed that the sulfhydryl-disulfide groups are not the only sensitive sites in proteins, since chymotrypsinogen, a protein containing no detectable sulfhydryl group, was rapidly denatured in the present solvent ~ y s t e m . ~ Acknowledgments.-The authors are grateful to Mrs. J. O'Brien and 0. Griffith for their technical assistance. (22) T. P. King, D. A. Yphantis and I,. C. Craig, THIS JOURNAL, 82, 3355 ( 1 Q U O ) .
LABORATORIES O F THEROCKEFELLER INSTITUTE,
S S l V I'ORK,
hT. Y.]
Distribution Studies with Human Plasma Albumin1 BY T. P. KING, D. A. YPHANTIS AND L. C. CRAIG RECEIVED DECEMBER 11, 1959 Dissolution of human plasma nlbumiii in a solvent system composed of ethanol, 1-propanol, water and ammonium sulfate leads t o the formation of a mixture of dimer and monomer. The two fractions can be separated by countercurrent distribution in this solvent system. The dimer was shown to be formed through an oxidative coupling of two molecules of mercaptalbumin.
Introduction In the preceding paper2 concerning the distribution of crystalline bovine plasma albumin, i t was (1) This investigation was supported in part by a research grant, A-2493 B.B.C., from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, Public Health Service. (2) T. P.King, D. A. Yphantis and L. C. Craig, THIS J O U R N A L , 82,
3350 (19GO).
reported that the albumin slowly underwent dinier and polymer formation when dissolved in a solvent system of ethanol, n-propanol, water and ammonium sulfate and that the reaction rate was diminished by the presence of sodium caprylate in the solvent system. The present paper will deal with the fractionation of human albumin in the same solvent system.
3356
T. P. KING,D. A. YPHANTIS9 N D L.
-
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Vol. s2
Lower
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X traveling 0.26 0.60
o'D. 280 mp 0.601-
0 60-
0.40-
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50
Fig. 1.-Distribution
$
46
I
100 150 Tube n u m b e p .
I
I
200
250
of human plasma albumin, 342 transfers.
In contrast to crystalline bovine plasma albumin, a portion of the human plasma aIbumin was found to undergo rapid dimerization in the solvent system, and sodium caprylate had no effect on the rate or extent of the reaction in this case. Upon distribution, an effective separation of the monomeric and dimeric albumins was easiIy realized due to the large difference in their partition coefficient values ( K ) . The formation of human plasma albumin dimer has been reported on two previous occasions. a Cohn, Hughes and WeareZanoted that in the process of removing adsorbed lipids from albumin by extraction with methanol or ethanol about 30% of the albumin was converted to the dimer. Straessle3b found that the treatment of the mercury dimer of mercaptalbumin with iodine effected a 60% conversion to the dimer. Experimental The human mercaptalbumin and an albumin preparation with a low sulfhydryl content which was a residue from the fractionation of mercaptalbumin from Fraction V were gifts of Dr. J. L. Oncley. The mercaptalbumin sample had 0.90 i 0.04 mole of sulfhydryl group by amperometric titration and ultracentrifugal analysis revealed its composition as 91 i 37, monomer and 8 It 370 dimer. The frac0.02 mole of tion with a low sulfhydryl content had 0.20 sulfhydryl group. Sulfhydryl group determinations, ultracentrifugal analyses and rotatory dispersions were obtained as in the preceding paper. F a t t y acid analyses were made bl- the method of Gordon . 4 The lyophilized albumin (30 mg.) was extracted with 15 ml. of a mixture of equal parts of iso-octane and glacial acetic acid for 30 minutes a t room temperature with occasional trituration. After decantation, the mixed solvent was washed with three 10-ml. portions of 0.1 hrsulfuric acid. A 3-ml. aliquot of the iso-octane extract plus 1 ml. of O.OOl:l, phenol red in 80% ethanol was then titrated under a fine stream of nitrogen gas with 0.01 N sodium hydroxide, using a microburet. With appropriate blank determinations the recovery was generally- 95r0 for a n amount of stearic acid similar to the acid content found in the albumin. Human plasma albumin was prepared by ammonium sulfate precipitation between 0.55 and 0.75 saturation. Blood from a single donor was withdrawn in a heparinized syringe. d f t e r removal of the cells by centrifugation at 5 " , the plasma (3) ; a ) E. J. Cohn, W. L. Hughes, Jr., and J. H. \Tear?, THIS ( b ) I
