Reports of the Academy of Sciences of the USSR

1. Volume 133, No. 2

CHEMISTRY

M. M. Botvinnik, A. P. Andreeva

Interaction of N-Benzoyl-O-(benzoylphenylalanine-C¹⁴)-serine with Ribonuclease

(Presented by Academician A. N. Nesmeyanov, 22 January 1960)

In our previous communication (¹) it was shown that N-benzoyl-O-(benzoylphenylalanine-C¹⁴)-serine

\[ \begin{array}{c} \mathrm{CH_2 - CH - COOH}\\ \quad | \qquad |\\ \mathrm{O} \qquad \mathrm{NHCOC_6H_5}\\ \quad |\\ \mathrm{C^*OCH(CH_2C_6H_5)NHCOC_6H_5} \end{array} \]

reacts in the presence of chymotrypsin with insulin and blood-serum albumin with transfer of benzoylphenylalanine-C¹⁴ to the protein.

In the present work an analogous study was carried out with ribonuclease. The reaction was conducted under the same conditions as before. Experimen-

Table 1

Interaction of N-benzoyl-O-(benzoylphenylalanine-C¹⁴)-serine with ribonuclease

* Pulses are given with background taken into account.
** Molecular weight of ribonuclease 13500.

mental data are presented in Table 1. As in the preceding experiments, treatment with alkali does not remove the label from the protein. However, in contrast to the behavior of other proteins, addition of chymotrypsin to the reaction mixture did not activate the process of transfer of benzoylphenylalanine onto the protein, but in most cases inhibited it. This course of the reaction led to the assumption that ribonuclease is capable of activating the process by itself, while chymotrypsin in this reaction is a kind of inhibitor. These assumptions were confirmed by the following experiments. In experiments 10 and 10a (Table 1), chymotrypsin inactivated by boiling was used. As a result, the number of imp/min per 5 mg of protein increased from 396 to 614–686. In experiments 12, 13, and 14 (Table 2), ribonuclease inactivated by oxidation was used.

Table 2

Incorporation of label into oxidized RNase and RNase treated with urea

Under the action of the O-peptide, incorporation of the label almost did not occur. If, however, chymotrypsin is added to oxidized ribonuclease and the O-peptide, a sharp increase in the labeling of the protein is observed (experiments 12 and 13, Table 2).

Oxidation of ribonuclease was carried out with performic acid ($^2$). Preparation “a” was precipitated with trichloroacetic acid, preparation “b” with acetone, and preparation “c” by lyophilization. In experiment No. 15, ribonuclease treated with urea was used. According to the literature, ribonuclease is only slightly inactivated by urea ($^3$), which was also reflected in the results. Ribonuclease oxidized with performic acid completely lost its ability to react with the O-peptide, while that treated with urea did so only partially (Table 2). The question of the nature of the bond between benzoylphenylalanine and ribonuclease requires further investigation.

Experimental Part

Crystalline ribonuclease (Hungary) was used in the experiments. The preparation was electrophoretically homogeneous. The interaction of ribonuclease with the O-peptide was carried out under the same conditions as previously ($^1$). The electrophoretic mobility of the preparation did not change after incubation.

Oxidation of ribonuclease. 1. Preparation “a” ($^{2,3}$). To a solution of 80 mg of ribonuclease in 0.8 ml of 98% performic acid was add-

add 0.02 ml of 30% $\mathrm{H_2O_2}$. After 30 min, the protein is precipitated with a tenfold volume of 10% trichloroacetic acid; the precipitate is centrifuged, washed with 5% trichloroacetic acid, with alcohol and ether (1 : 3), with ether, and dried.

1. Preparation “b”$^{(2)}$. Oxidation is carried out analogously to that described. The protein is precipitated with acetone; the precipitate is centrifuged, washed with acetone, and dried.

2. Preparation “v”$^{(3)}$. 0.45 ml of 30% $\mathrm{H_2O_2}$ is added to 9 ml of 98% formic acid and left for 1 hour at $20^\circ$; then the mixture is cooled to $0^\circ$. 5 ml of the cooled solution is added to 100 mg of ribonuclease in 5 ml of formic acid. After 2 hours (with cooling), the mixture is diluted with a tenfold volume of water and lyophilized.

Treatment of ribonuclease with urea$^{(4)}$. Crystalline urea is added to the reaction mixture (O-peptide and ribonuclease) to bring the solution to 8 M concentration, and incubation is carried out under the same conditions as before.

Thus, it has been shown that N-benzoyl-O-(benzoylphenylalanyl-$\mathrm{C}^{14}$)-serine reacts with ribonuclease with transfer of benzoylphenylalanine to the protein. The reaction is inactivated by chymotrypsin.

We express our gratitude to M. G. Kritsman and A. S. Konnikova for assistance in the work, and to M. A. Prokof’ev for providing the ribonuclease.

Moscow State University
named after M. V. Lomonosov

Received
10 II 1960

References

1. M. M. Botvinnik, A. P. Andreeva, DAN, 133, No. 1 (1960).
2. C. Hirs, J. Biol. Chem., 219, 611 (1956).
3. W. P. Harington, S. A. Schellman, C. R. trav. Lab., Carlsberg, 30, 21 (1956).
4. C. B. Anfinsen, ibid., 30, 13 (1956).