3802
COMMUNICATIONS TO
indicates that the complexes are formed in a 1:1 ratio. Values of the association constant, K , and of the molar extinction coefficient e are reported in Table I. The spectra of these change transfer complexes are similar in both contour and intensity to the spectrum of GPD in presence of DPN.6-9 This suggests that in the GPD-DPN complex the pyridinium moiety of the coenzyme molecule interacts with indole side chains of the enzymelo; this interaction should almost certainly occur in the case of GPD-APDPN complex, since iodoacetate only slightly reduces the absorption in the 360 mp r e g i ~ n . ~ Complexing said to be associated with electron transfer from the indole nucleus to flavines and pteridines has been reported recently.I1 Acknowledgment.--We are indebted to the Rockefeller Foundation and to the Brazilian “Conselho Nacional de Pesquisas” for grants, and to the latter also for a fellowship granted to P. G. (6) E. Racker a n d I. K. Krimsky, J . B i d . Chem., 198, 731 (1952). (7) S. F. Velick, ibid., 203, 563 (1953); in “Mechanism of Enzyme Action,” W. D. McElroy and B . Glass, eds., T h e John Hopkins Press, Baltimore, hfd., 1954, p. 500-505. (8) J. B. Fox, Jr., and W. B. Dandliker, J . Biol. Chem., 221, 1005 (1956). (9) N. 0. Kaplan, M. M. Ciotti and F. E. Stolzenbach, Archiu. Biochem. B i o g h y s . , 69, 441 (19573. (10) It is true t h a t -SI3 reagents abolish t h e 305 m).i interaction in the D P D - D P N complex (ref, 6-8 and also B. Chance in ”Mechanism of Enzyme Action,” W. D . McElroy and B. Glass, eds., T h e Johns Hopkins Press, Baltimore, Md., 1954, p. 434-438), b u t as pointed o u t by Chance and by Velick (ref. 7) this does not necessarily indicate a direct binding of coenzyme t o sulfur. (11) H . A. Harbury and K. X.Foley, Pvoc. S n l l . Acnd. S c i . , 4 4 , 662 (1958); I. Isenberg and A. Szent-Gyorgl-i, i b i d . , 44, 857 (1958); E. Fujimori, i b i d . , 45, 133 (1959); R . Pullman and A. Pullman, ibid.,44, 1197 (1958); 45, 136 (1959).
THE
EDITOR
Vol. 81
S
/ \
c=o
II?C
I
H r C-CH
+ H2S-protc.iri -+
1
I
~HCOCH~ HS-CHaCH&HCO--NH--protein
AHCOCH~
(2)
Although direct reaction is not especially useful, with Agf as adjuvant3 thiolation of gelatins has been effected rapidly a t p H 7.5. However, the presence of silver, and subsequently 1 M thiourea for its removal, presents problems particularly with disulfide-containing proteins. These problems are avoided by a suitable extension of the reaction of acid anhydrides with proteins6 For example with S-acetylmercaptosuccinic anhydride 0
CH ,CO-S-C€I-C/
CH?CO-S-CIICO-SH-prot
ein
I
(IT)
+
CHjCOOH ~~S-C€€CO-~I~-I)r~)teiii (3) (111) &H.COOH
Solid anhydride6 is added to protein solution at, for example, fiH 7, over from 0.25-1 hour, depending on the amount of reagent. The p H is maintained by adding sodium hydroxide. Air is excluded throughout with nitrogen. (Coupling can be performed over a range of p H and temperature.) Hydrolyzed anhydride (I) is removed with an anion exchanger, salts with a mixed-bed exchanger DEPARTMENT OF CHEMISTRY or by dialysis. The mercaptosuccinylated proFACULDADE DE FILOSOFIA, CIENCIAS E LETRAS DE SXo PACLO GIUSEPPECILENTO tein is isolated by lyophilization. UNIVERSIDADE S.Xo PAULO,BRASIL PAOLAGICSTI Typical results at room temperature are sum RECEIVED ?IIIAY (5, 1959 inarized in Table I. TABLE I MERCAPTOSUCCINYL PROTEINS
A NEW METHOD FOR THE INTRODUCTION OF THIOL GROUPS INTO PROTEINS
hloles (I)
Iaaea _____
Sir: For fundamental and technical reasons many attempts have been made to introduce sulfur de novo into proteins. Using thioglycolides, Schoberl’ prepared highly thiolated casein and ovalbumin (-sCHYCO-sCH2CO-),
+ HzN>protein H2N I-IS-CHzCO-NH HS-CH3CO-NH
-+-
)protein
(1)
Since Schoberl’s reagent is difficult to characterize, the recent method of Benesch and Benesch2J using N-acetylhomocysteine thiolactone is more attractive (1) A. Schoberl, Atzgew, Chem., 60, 7 (1948). (2) R . Benesch and R. E . Benesch, TITISJ O U R N A L , 78, 1597 (l!X6). (3) R. Benesch and R. E . Benesch, P i q c .Vall, A r a d . Sci.. 44, 848
(1958).
Protein
Gelatin Gelatin Gelatin Bovine serum albumin Bovine serum albumin Ribonuclease
per 106 grams of protein
30 120 360 45 360 140
Moles introduced per 10s grams of protein Acetyl\fermer~... captocaptop H of succinyl succinyl reactlon (111) (11) ~~~
7
6
8 8
12
8 8 7
14 5 8 2
12 17 23 21
54 6
A reaction analogous to ( 3 ) giving mercaptosuccinylated esters has been achieved with polyhydroxylic molecules (e.g., dextran, polyvinyl alcohol). (4) It has been successfully applied a t high $11 (10.7) b y S. J . Singer, J. E . Fothergill and J. R. Shainoff, THISJ O U R N A L , 81, 2277 (1969). (5) H. Frankel-Conrat, R . S . Bean and H. Lineweaver, J . Biol. Chem., 177, 385 (1949); P . H. Maurer and H . I,ebovitz, J . Immuizol., 76, 335 (1956); A. F. S. A. Habeeb, H . G. Cassidy and S. J. Singer, Biochim. Biophys. Acta, 29, 687 (1958). (6) B. Holmberg and E. SchjBnberg, Arkiv Kemi Mineual. Geol., 1 4 8 , No. 7 (1940).
July 20, 1959
3803
COMMUNICATIONS TO THE EDITOR
T h a t the mercapto groups are really bound t o protein, and not merely the result of inefficient separation, was shown by ultracentrifugation with a colored azomercurial. At 60,000 r.p.m., the schlieren and color boundaries moved together. The a c e t y l 3 linkage of (11) is stable for days in aqueous solution a t PH’s as high as 9.5. Conversion of (11) into (111), if desired, can be accomplished in a few minutes in dilute sodium hydroxide, p H 11.5. Preliminary experiments indicate that some nitrogenous bases also work a t pH’s much nearer 7 (e.g., imidazole).
TABLE I RADIOACTIVE PRODUCTS OF THE GASEOUS REACTION OF ENERGETIC TRITIUM ATOMSWITH CYCLOPROPANE Gas Pressure, cm. Irradiation conditions n./ cm.l/sec. Product
A
HT CH,T
c-c
c=c DEPARTMENT OF CHEMISTRY NORTHWESTERN UNIVERSITY IRVING M. KLOTZ c-c-c EVANSTON, ILLINOIS RICHARD E. HEINEY c-c=c RECEIVED MAY25, 1959 C HIGH ENERGY EXCHANGE REACTION OF TRITIUM ATOMS WITH CYCLOPROPANE
Sir: Our recent experiments with tritium atoms slowing down from very high energies in the presence of cyclopropane show a substantial incorporation into the organic molecule by reaction (l),in which the asterisk designates an energetic species
)c-c c-c-c-c
C c>c-.
16.4 47.4 6.8 4.7 1.8 6.5 2.5
15.3 58.3 6.0 4.0 1.8 4.1 2.2
10.9 30.1 1.8 7.8 2.0 13.7 1.8
7.4 54.9 6.8 6.4 2.0 6.8 1.7
3.0
1.5
1.0
3.1
1.8
Low 8’6
}4.9
}1.8
13’* Low
}5.0
5.4
4.0
1.5
7.8
3.7
1.4
1.7
0.7
0.7
0.5
0.1
1.8 0.5
