Oct. 5 , 1957
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COMMUNICATIONS TO THE EDITOR
of amino acid provide a rational basis for the interpretation of patterns which may be attributed t o proteins in solution. (5) National Research Council Fellow. (6) American Cancer Society Fellow.
CONTRIBUTION No. 2238 GATESAND CRELLINLABORATORIES OF CHEMISTRY CALIFORNIA INSTITUTE O F TECHNOLOGY OLEG JARDETZKY6 PASADENA 4, CALIFORNIA CHRISTINED. J A R D E T Z K Y ~ RECEIVED AUGUST19, 1957 THE ROLE OF DISULFIDE BONDS IN ANTIBODY SPECIFICITY Sir :
TABLE I EFFECT OF DISULFIDEREDUCTION O N ANTIBODYBINDING^ Expt.
Protein
Treatment
I
Y/C
x
lo-'
0.74 14.4 Dialysis A Anti-Lac .62 7.0 Detergent A Anti-Lac .70 5.6 Anti-Lac Detergent B .22 1.5 Anti-Lac Reduction B .13 0.8 Anti-L-I, Reduction B B Anti-L-I, Detergent .06 .3 B Anti-L-I, ....... . 00 .o B RY *G Reduction .14 .9 M 0 One ml. of protein solution approximately 2 X was dialyzed against 1 ml. of 4 X 10-6 M dye solution. The -SH content of the reduced proteins was measured by the amperometric titration method of Benesch, et aZ.* Different anti-Lac preparations were used in experiments A and B.
The occurrence of disulfide bonds in y-globulins3p4and antibody proteins6 has led us t o suggest6that these linkages play an essential role in the evident that detergent treatment alone reduces the maintenance of the specific configuration of the specific binding somewhat, about 2-fold, and that combining region of the antibody molecule. We the capacity for non-specific binding is acquired by have obtained evidence supporting this hypothesis the reduced proteins. When these effects are from experiments in which the disulfide bonds of taken into account the results demonstrate that the purified anti-hapten rabbit antibodies have been specific binding is greatly reduced, to the extent of reduced and the resulting sulfhydryl groups pre- about 7-fold, when reduction of the disulfide bonds vented from re-oxidizing. The effect of such re- occurs. The residual specific binding observed duction on the specific combination of the antibody may be due t o the fact that only about 10 disulfide with an homologous azohapten provided the basis bridges, out of a minimum content of 20,5 were for our conclusions. split in our procedure. The protein was reduced with 0.1 M P-rnercaptoThe additional negative charge acquired by the ethylamine-HC1 a t $H 7.4 in the presence of reduced antibody by reaction of the -SH groups 0.1 Jf sodium decyl sulfate. The reducing agent with iodoacetate probably does not play a major role was removed by passage of the reaction mixture in the reduction of specific binding. Reaction of through a column of Dowex 50-X8(Na+). The reduced antibody with iodoacetamide shows the effluent reacted with an excess of iodoacetate by same results but such a preparation is insoluble a t overnight stirring a t room temperature. These p H 7.4 and is therefore less useful than the iodoaceoperations were done under anaerobic conditions tate derivative. by a procedure which will be described in detail R. E. Benesch, H. A. Lardy and R. Benesch, J . B i d . Ckem., 216, elsewhere. The protein solutions were subjected 663(8)(1955). to exhaustive dialysis against 0.001 M phosphate DEPARTMENT OF PEDIATRICS buffer p H 7.4 for the removal of the detergent. SCHOOL OF MEDICINE FREDKARUSH The low ionic strength was necessary to avoid the UNIVERSITYOF PENNSYLVANIA AND THE precipitation of the protein derivative a t this pH. CHILDRES'SHOSPITALOF PHILADELPHIA PA. The test antibody was that specific for the p- PHILADELPHIA, RECEIVED AUGUST21, 1957 azophenyl @-lactoside group (anti-Lac)' and the control proteins were rabbit y-pseudoglobulin (RypG) and antibody specific for the L-phenylDETERMINATION OF THE SITE OF I4C IN (p-azobenzoylamino)-acetate group (anti-L-I,) .6 HYDROCORTISONE-14C DERIVED FROM The ability of the reduced proteins and various CHOLESTEROL-21-'4C INCUBATED WITH BOVINE ADRENAL GLAND TISSUE' control preparations to bind the azohapten 9-(pdimethy1aminobenzeneazo)-phenylP-lactoside (Lac Sir: dye) was measured by equilibrium dialysis a t 25' The biochemical conversion of cholesterol to the in 0.001 M phosphate buffer, p H 7.4. The results CP1-steroidsof the adrenal cortex was first demonare shown in Table I in terms of 7 and Y / C where r strated by investigators2 using cholester01-3-~~C. is the average number of dye molecules bound per Later other workers3 reported the isolation of a protein molecule a t the free dye concentration c. labeled six-carbon fragment resulting from the The last column, headed r / c , provides the most biochemical degradation of cholester01-26-~~Cby useful measure of the binding affinities. It is mammalian tissue extracts. The latter study suggested that the steroid hormones of the adrenal (1) These studies were aided by a grant from the National Science cortex could be derived from cholesterol involving Foundation and by a research grant (H-869) from the National Heart Institute of the National Institute of Health, Public Health Service. a degradation of the last six carbon atoms of the (2) I am indebted to Mrs. F. Karush for technical assistance in this side chain [C,r.C,l i(Cd. I. investigation, To obtain further experimental evidence on the (3) E. L. Smith and B. V. Jager, Ann. Rev. Microbiol., 6, 214 (1952). (4) 0. Markus and F. Karush, THISJOURNAL, 79, 134 (1957). (5) E. L. Smith, M. L. McFadden, A. Stockell and V. BuettnerJanusch, J . B i d . Chem., air, 197 (1955). (6) F. Karush, THISJOURNAL,18, 5519 (1966). (7) F. Karush, ;bid,, 79, 3380 (1967).
(1) This investigation was supported by the John J. Morton Cancer Fund and by a fellowship (HF-6137) from the National Heart Institute of the Public Health Service. (2) A. Zaffaroni, 0. Hechter and G. Pincus, THISJOURNAL, 73, 1390 (1951). (3) W.S,Lynn, Jr., E,Staple and S. Gurin, i b i d . , 76, 4048 (1964).
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COMMUNICATIONS TO THE EDITOR
Vol. 79
pathway of the biochemical conversion of choles- possible to perform differential experiments terol t o hydrocortisone, cholester01-2I-'~C was whereby very small changes in sedimentation coprepared4 and gave, on incubation with bovine efficients can be measured accurately. This comadrenal gland tissue, h y d r o c ~ r t i s o n e - ~ .5 ~ C h part munication deals with such experiments and some of the isolated h y d r o c ~ r t i s o n e - ~ ~(5.15 C mg.; potential applications. specific activity, 159 d.p.m./mg.) was diluted with The technique involves a comparison, a t conan equal amount of non-labeled hydrocortisone, jugate levels, of the refractive indices of two soluand then degraded by periodate oxidation according tions contained in separate compartments of a to a procedure previously employed for similar double-sector ultracentrifuge cell. If both solureactions.6 From the oxidation mixture, 7.5 mg. tions are identical the Rayleigh pattern consists of of neutral material was recovered and was found a series of parallel, straight interference fringes. t o contain no hydrocortisone after paper chroma- For the fringes to be straight even in the presence tographic analysis. From the acidic fraction of concentration gradients, the two boundaries (9.7 mg.), 1lp, 17a-dihydroxy-3-oxo-4-etiocholenicmust migrate and spread a t the same rate so that acid7 was isolated and identified by comparison the refractive index difference a t conjugate levels with an authentic sample, by mixed paper chroma- throughout the cell is constant. When one boundtography,s and by its ultraviolet absorption spec- ary moves faster than the other, the fringe pattern trum in concentrated sulfuric a c d g This etio a t the boundaries is warped to produce curved acid was found t o contain no radioactivity.'O fringes whose shape resembles the tracings proCarbon atom 21 of the hydocortisone-lK! was duced by schlieren optical systems. Depending obtained as formaldehyde which was isolated as upon which compartment contains the faster its dimedone derivative, 4.6 mg., m.p. 191.5-102°. moving species, the pattern will show a maximum A mixture of this derivative and an authentic or a minimum. n'ith transport equations the reference sample (m.p. 192-193') melted a t 191.3- difference in sedimentation coefficients can be 192' (melting points not corrected). The dime- expressed directly in terms of the change with time done derivative had a radioactivity'O of 2G of the first moment of the area defined by the curved d.p.m./mg.ll fringes. This evidence demonstrates that carbon atom 21 T o rest the method small amounts of DnO were of cholesterol is maintained during the biochemical added to different solutions of bushy stunt virus, conversion of cholesterol to hydrocortisone and that and the reduction in sedimentation coefficient of the biosynthesis of hydrocortisone from cholesterol the virus was measured by the differential techin the adrenal cortex involves the elimination of nique. For decreases of O.82YO and l,65y0 (detercarbon atoms 22-27 of the original cholesterol side mined by interpalation of data from conventional chain. ultracentrifuge gives 0.7SYO and 1.61YO, respec(4) P. Kurath and M. Capezzuto, THIS JOURNAL, 78, 3527 (195G). tively. Comparable precision is realized in experi( 5 ) F. M. Ganis, P. Kurath and &I. Radakovich, F e d e r d i o i t Proc., ments with proteins. 16, 357 (1957). The sensitivity and accuracy of the method even (6) S. A. Simpson, J. F. Tait, A. Wettstein, R. PJeher, J. v. Euw, a t this early stage of development are sufficient 0. Scbindler and T. Reichstein, H e l v . Chim. Acta, 37, 1200 (1954). (7) J. v. Euw and T. Reichstein, ;bid,, 26, 988 (1942); H. L. hlason, to commend it for many experiments involving W.31.Hoehn and E. C. Kendall, J. Biol. Chem., 124, 459 (1938). small differences in sedimentation coefficients. (8) A. Zaffaroni and R. B. Burton, ibid., 193, 749 (1951). These changes may result from reduction in the (9) A. Zaffaroni, THISJOURNAL, 72, 3828 (1950). buoyancy term as with DzO or from a change in the (10) Radioactivity determinations by New England Nuclear Corporation, Boston, Massachusetts. molecular weight or frictional coefficient of the (11) The amount of the dimedone derivative obtained was in excess macromolecule. The latter is illustrated by preof the expected amount and the radioactivity of the sample was lower liminary results from a study of the binding of than the calculatedvalueof 98 d.p.m./mg. However, it was found that small ions to serum albumin, in which the sedimenthe elution of two filter paper blanks yielded 23.2 and 34.3 mg. of residue which gave upon analogous periodate oxidations and reaction with tation coefficient decreased by about 0.5y0despite dimedone, 2.4 and 3.5 mg. respectively of the dimedone derivative of an increase of 4YO in molecular weight. The techformaldehyde. This would account for t h e lower radioactivity obnique also provides valuable data for the analysis tained in the experimental sample. of equilibrium systems involving rapid reactions DEPARTMENTS OF SURGERY (DIVISIONOF CANCERRESEARCH) P. KURATH between a small ion, A, and a protein, P, to form AND BIOCHEMISTRY, UNIVERSITY OF F. M. GAXIS complexes, PAi, with i varying from 0 to n. For ROCHESTER MEDICALCENTER M. RADAKOVICHsuch systems equation (1) gives the equilibriuni ROCHESTER 20, NEW PORK concentration of free A component RECEIVED AUGUST5 , 1957
A DIFFERENTIAL ULTRACENTRIFUGE TECHNIQUE FOR MEASURING SMALL CHANGES I N SEDIMENTATION COEFFICIENTS'
( 1 ~
[A]and [?I
are the total concentrations and SA and
Sir:
sp are the sedimentation coefficients of the pure
With the recent adaptation of the Rayleigh interferometer to the ultracentrifuge,* i t has become
components, and SA and Sp are the constituent
(1) This work has been supported in part by a grant from the National Science Foundation. (2) This optical system is available commercially from the Spinco Division, Beckman Instruments, Inc., Palo Alto. California.
(3) Svenssona obtained similar fringe patterns with the Rayleigh interferometer during examination of two identical diffusing boundaries which were slightly displaced from one another. (4) H. Svensson, Acta Cheiit Scnnd , 3, 1170 (1049). ( 5 ) H.K. Schachman. in preparation.
