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Vol. 79
aldose which failed to react with periodate. These findings along with published datal permit the assignment of the structure of methyl 2-0-carbamyl-4-0-methyl-5,5-dimethyl-~-lyxoside to 111 and show that I1 is the 2-carbamyl analog of 1.
tyrosine hydrogens, as suggested. l I n aqueous solutions these shifted - 20 to - 30 C.P.S. relative to the aromatic proton of toluene as 0. I t probably also includes the 8 CH hydrogens on the imidazole of histidine, found a t somewhat lower fields. The total number of non-exchangeable hydrogens in the CH3 protein being 697,4this will account for S.970of the CH3COCI Me0 k 0 total area under the absorption curve, in agree:I -A MeOH A’: ment with the 9 =t1% measured. IVhile in water the guanidino group of arginine gives rise to an HO OCONHz additional peak in the same region, this disappears after equilibration with D,O, so that it would not likely contribute to peak I of ribonuclease in this Me0 0 instance. H ,OMe MH eFGH,OH Y“ A second peak, due to the 123 wCH protons, of 1 1 HO O C O N H z HO OH all amino acids the 30 @-protons of serine and 10 @-protonsof threonine should be observed at +90 YI v to +I10 C.P.S. relative to toluene if no shifts due to peptide bond formation were to be expected. I n addition, the 6 glycine protons and 8 protons of the CH, group in arginine are observable at slightly OH HO higher fields of +I17 to +I20 C.P.S. in a region CH3 intermediate between the second and third peaks. 1Y Together with the protons of the second peak they It appears, therefore, that the antibiotic activity would account for 23.47,of the total area, as com2%. of novobiocin is highly dependent on the presence pared t o the experimental estimate of 26 As peptide bond formation causes a displacement of and location of the carbamate group. We gratefully acknowledge the contributions of a-CH peaks 15-20 cycles toward lower fields, the Drs. W. G. Jackson, W. T. Sokolski, J. L. Johnson, maximum of the second peak of ribonuclease, should appear at about +SO to +90 c.p s. where it Mrs. G. S. Fonken, and Mr. W. A. Struck. is actually found. RESEARCH LABORATORIES J. W. HINMAN A third peak with a maximum between 13.5 to THEUPJOHNCOMPANY E. L. CARON KALAMAZOO, MICHIGAS H. HOEKSEMA +145 C.P.S. is accounted for by CH2 groups as follom-s: 16 from cysteine-cystine, 8 from methRECEIVED AVGUST8, 1957 ionine, 32 from aspartic acid, 20 from lysine ( E -CH2), 10 from proline, 8 from histidine, 6 from phenylalanine and 12 from tyrosine. This amounts AN INTERPRETATION OF THE PROTON MAGNETIC t o 15.27, of the total area, the experimental estiRESONANCE SPECTRUM OF RIBONUCLEASE mate for the peak being 18 + 3c/,. The 64 CH2 Sir: groupsof glutamicacid fallat +156c.p.s., accounting Saunders, Wishnia and Kirkwoodl recently have for an additional !3,2YGof the area, which could be reported the first proton magnetic spectrum due to a attributed either t o the third or the fourth peak. protein, pancreatic ribonuclease, in D 2 0 . The It is probable that peak I V is actually a fusion of spectrum consists of four broad peaks, falling two broad peaks, one with a maximum a t +180 to roughly into the range between the aromatic and the +190 c.P.s., the other a t $210 to $220. The aliphatic peaks of toluene. The authors have first of these is due to aliphatic CH groups of identified the first peak tentatively, corresponding leucine (2 hydrogens) isoleucine (3) and valine t o lower field strength, as due to the aromatic (9), CH2 groups of leucine (4 hydrogens), isohydrogens and the fourth peak, a t highest field leucine (G), proline (20)) lysine (BO’, and arginine strength as due to “aliphatic carbon atoms attached (16) and the CH3 group of methionine (12 hydroonly t o other aliphatic carbons,” stating that a gens), accounting for 19.17, of the area. The complete interpretation is not possible a t present. second, due to CH3 groups of alanine (36 hydroHowever, our studies on the proton magnetic gens), valine (M),leucine (12), isoleucine (18),and resonance spectra of amino acids and peptidesz3 threonine (30) would respresent 21.6%. The together with the amino acid composition of combined area under peak IV. including glutamic ribonuclease determined by Hirs, Stein and Moore, acid is 49.9$,, closely in agreement with the experipermit the prediction of a complete n m . r . spec- mental value of 47 =k 37,. It would be of interest trum for the protein, which is in excellent agree- if it were possible to sufficiently average the field ment with experiment. seen by the aliphatic protons-perhaps a t higher There is little reason to doubt that peak 1 is temperature-to actually resolve the fourth peak. due essentially to the 18 phenylalanine and 36 It might also be noted, that due t o peptide bond formation small and somewhat variable shifts (1) XI Saunders A Wishnia a n d J G Kirkwood THISJ O U R V A L 79, 3289 (1957) toward lower fields can be expected for hydrogens (2) M Takeda end 0 Jardetzky, J Chem Phvs 26, 1346 (1957) contributing to peaks XI1 and I T T making :>nevact (3) 0 Jardetzky end C D Jardetzky, t o be publlshed prediction of their maximum dificult. However, (4) C H W Him, w k , Fltdbl and 9 M o o r e J BIdl Ckrm 911, 841 (1964) even the foregoing illustrates that I\TM R spectra
VHpMe
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+
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).
