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COMMUNICATIONS TO THE EDITOR
Sept. 5, 1958
TABLEI
Compound
XAD XRP’ XR’ x9
COMPOSITION AND CHROMATOGRAPHIC CHARACTERISTICS O F Ribose Phosphate mole/mole base mole/mole base 15 min. 75 min. 10 min. Total Isob
1.50 0.56
2.05 0.95 1.16 0.00 . 00 1.00
2.04;
0.10
.00 . 00 . 00
XAD
Etho
0.21 .21 0.40-. 45 .62 .go(,90)
1.02 0.00 .00 . 00
AND ITS
DERIVATIVES
R, in solvents”
Phd
0.10 .18 .62
Bute
0.55.65 .42-.49
0.41(.41) Adenine/’h . 00 6’-AMP 1.00 X derivatives were located with a Mineralight lamp (Max. emission 253 mp). Isobutyric acid, NHa, H20 (66:1:33). Ethanol, 1 M ammonium acetate pH 7.5 (7:3). Phenol, HsO ( 8 : 2 v:v). Butanol, acetic acid, 0 Obtained from XAD after H20 (4:1:5). f Obtained from XA? after 10 min. hydrolysis in 1 N HCl a t 100’. IniValues in parentheses correspond to adenine isolated from 5‘ -4MP. 1 hour hydrolysis with 1 N HCl a t 100 . tial inorganic phosphate was zero.
X passes from pH 6 to 12 the 366 mp band shifts trypsin, 5.77* for chymotrypsinogen3 and 7.8y0 to 405 mp (Am a t pH 12 = 11.2 X lo3). A t p H for l y s o ~ y m e . ~The use of differential ultraviolet above 9, X R P has another band with maximum a t spectrophotometric recording allows the detection 854mp (Am = 2.87 X lo3). 70 I: 1 I I The Am values for X R P were calculated assuming the ratio X : ribose (or X : phosphate) = 1: 1. Eri When these values were subtracted from the spec60 trum of XAD, the differential spectrum corresponded t o 1 mole of adenosine monophosphate t per mole of X R P (Am a t 257 mp for AMP in XAD, 50 calcd. = 14.9 X lo3; reference value3 for 5’ AMP Am = 15 X lo3). XAD was obtained from the Ba soluble fraction of rabbit muscle extract, which was absorbed on 40 Dowex 1 (formate form), eluted with 4 M formic I-0 acid and purified by paper chromatography and Q electrophoresis. The average yield was 1 pmole 30 z_ per kg. of muscle.
8
& 8
(3) R. M. Bock, et ol., Arch. Biochcm. and Biophys., 62, 253 (1956). BIOCHEMICAL SECTION OF THE W. HENRY MOSLEY
OKLAHOMA MEDICAL RESEARCH FOUNDATION OKLAHOMA CITY, OKLA. RANWEL CAPUTTO RECEIVED JUNE 17,1958
SELECTIVE CLEAVAGE OF PEPTIDE BONDS. 11. THE TRYPTOPHYL PEPTIDE BOND AND THE CLEAVAGE OF GLUCAGON1
Sir: When the action of N-bromosuccinimide on indole derivatives such as carbobenzyloxy (Cbz)tryptophan, acetyltryptophan, Cbz-tryptophylglycine and tryptophan-containing peptides and proteins] such as gramicidin D, chymotrypsin(ogen) and lysozyme, in dilute aqueous solutions (2 X iM> is followed i n situ with a self-recording ultraviolet spectrophotometer, one notices the disappearance of the indole absorption a t 280 mp and the concomitant appearance of a new band a t 240250 mp and a low-intensity band a t 307 mp. The effect of added N-bromosuccinimide on the indole spectrum is instantaneous and linear up to the consumption of ca. 1.5 moles/mole of tryptophan with optimal conditions a t PH 4 in aqueous acetate buffer. Multiplication of the decrease in optical density a t 280 mp by an empirical factor (1.31) gives the extinction due to tryptophan in the peptide or protein. The “titration” of tryptophan in representative proteins yielded 5.77$ for chymo(1) Presented in part before the Division of Biological Chemistry e t the 134th ACS Meeting in Chicago, Sept. 7-12, 1958.
W
v)
a w U
z
20
10
I
2
3
4
5
MOLES OF N-BROMOSUCCINIMIDE,
Fig 1. The liberation of glycine from N-benzoyltryptophylglycine ( O ) , indole-3-propionylglycine ( X ) and carbobenzyloxytryptophylglycine (0)as a function of the addition of N-bromosuccinimide to the solution of the peptides in acetate formate buffer a t p H 4. The decrease in optical density a t 280 mp ( . . . . .) reaches 100% after the addition of 1.53 moles of NBS.
and determination of tryptophan bound in protein on a micro scale and offers advantages over known spectral methods.6 After determination of the tryptophan content (2) Reported 5.7%: J. L. Weil and A. R . Buchert, Arch. Biorhcm. Biophys., 46, 266 (1953). (3) Reported 5.6%: cited in J. H. Northrop, M. Kunitz and R. M. Herriott, “Crystalline Enzymes,” 2nd Ed., Columbia ‘C‘niversity Press, 1948, p. 26. New York, N. Y., (4) Reported 7.1 and 9.1%: C.Fromageot and M. Privat de Garilhe, Biochim. et Biophys. Acto, 4, 509 (1950), and J. C . Lewis, N. S. Snell, D. J. Hirschmann and H. Fraenkel-Conrat, J. Bid. Chon.. 186, 23 (1950). C j . t h e decrease of c a t 280 ma in the oxidation of lysozyme with periodate: K. Maekaws and M. Rushibe, Bull. Agricultural Chcmical Society o f J o p a n , 19, 28 (1955). (5) Cf.W. L. Bencze and X.Schmid, A n a l . Chrm., 39, 1193 (1957).
COMMUNICATIONS TO THE EDITOR
4748
of a peptide or protein by N-bromosuccinimide "titration," an additional 1-2 moles of N-bromosuccinimide per mole of tryptophan is added for the controlled cleavages of the C-tryptophyl bonds. Figure 1 summarizes experiments with model peptides. The general usefulness of the method was demonstrated with g l ~ c a g o n , ~ the ~ *crystalline hyperglycemic-glycogenolytic peptide from pancreas, containing only one tryptophan among 29 amino a ~ i d s . ~ N-Bromosuccinimide leads t o the liberation of a major new ninhydrin-positive peptide, giving positive platinic chloride reaction for methionine'O and negative reactions for histidine and arginine. Its hydrolysis yielded aspartic acid, threonine, methionine and leucine. This tetrapeptide, which arises from the C-terminal sequence TRY-LEU-MET-ASP-THR, has been obtained by the action of chymotrypsin" and trypsin'2 on glucagon. However, the cleavage of glucagon by N-bromosuccinimide is more rapid (
