Effect of some solvents on the enzymic activity of ribonuclease

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Effect of

solvents on the enzymic activity of ribonuclease

some

The effect of solvents on the secondary structure of RNAase was studied by YAtCG AND Dol-Y1 and by WEBB AND TANFORD2. From the changes in optical rotation it was assumed that in water medium the molecule is only partially folded whereas in solvents such as N,N'-dimethylformamide and 2-chloroethanol the folding of the protein increases. Previously we have studied the effect of dimethylformamide on the activity of several enzymes 3, 4. It was found that the activity of the intracellular enzymes tested (e.g. aldolase, D-glyceraldehyde-3-phosphate dehydrogenase) increased while that of the extracellular proteases (e.g. pepsin, trypsin, chymotrypsin) was inhibited as the concentration of dimethylformamide was increased from o to approx. 30 %. This report presents some data on the influence of solvents as dimethylformamide, chloroethanol, dioxane, glycerol, methanol, ethanol, n-propanol and n-butanol on the RNAase activity. Ribonuclease isolated from bovine pancreas according to McDONALD5 was recrystallized three times with ammonium sulfate and three times with alcohol. Samples containing 20 ~g enzyme/ml were preincubated with increasing amounts of solvents (0-50 %, v/v) in 0.5 M acetate buffer (pH 5.0) and tested for RNAase activity. The time of incubation between o and 60 min did not influence in any way the results obtained. The assay system contained o.I ml enzyme, 2 mg RNA (from yeast, Merck, purified according to WOODWARD6) and solvent in the same concentration as in the preincubation mixture, in a final volume of I.O ml. After 5 rain incubation at 25 ° the reaction was stopped by addition of 5 % trichloroacetic acid plus 0.25 % uranyl acetate and centrifuged. The absorbancy of the supernatant was measured at 260 m y against a blank containing all the ingredients except the enzyme. Fig. I represents the effect of different solvents on the RNAase activity. It can be seen that up to 35 % concentration both dimethylformamide and chloroethano] increase the enzyme activity more than twice. 50 % chloroethanol causes a significant inhibition whereas even in the presence of 50 % dimethylformamide the activity does not decrease below the original value. The effect of dioxane in the range of 0-20 %

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Fig. I. RNAase activity in presence of different solvents. Solvents: i, dimethylformamide; 2, 2-chloroethanol;3, dioxane; 4, glycerol; 5, methanol; 6, ethanol; 7, n-propanol; 8,n-butanol. Biochim. Biophys. Acta, 53 (1961) 218-219

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is practically negligible. In the presence of 35 % dioxane the RNAase activity is approx. 40 % higher than in the control sample, but more than 40 °/o dioxane causes inhibition. No increase in the activity was observed in the presence of glycerol. An interesting phenomenon was observed when RNAase was preincubated with alcohols. It can be seen on Fig. I that the effect of alcohols depends on their chain length. Methanol causes an increasing inhibition. The effect of ethanol is similar but relatively smaller. Propanol in 0-30 % concentration has hardly any effect on the enzyme activity, and causes only in higher concentration a slight inhibition. Butanol up to 20 O,o concentration increases the activity. The effect of butanol in higher concentrations could not be investigated because it is not soluble under these conditions. From the above data we conclude that the effect of solvents on the enzyme activity is not so much connected with the decrease ill water concentration as with the increase of the non-polar character of the medium. It can be said that there m a y be a close correlation between the secondary structure and enzyme activity, namely conditions favouring a small increase in folding of the molecule 1, 2 m a y increase the enzyme activity too. FINDLAY et al. 7 have reported some analogous data. They have studied the influence of dioxane on the RNAase activity in methanol-water mixture and have found that under their experimental conditions the rate of the hydrolytic activity increased, while that of the esterolytic activity decreased. From the physiological point of view one m a y regard RNAase either as an extraor as an intra-cellular enzyme. This duality is also reflected in its properties. Its molecular weight is low, it is cross-linked b y m a n y disulfide bridges and is partially folded in aqueous solution, that is to say, from the point of view of its structure, it is similar to the secretory pancreatic enzymes (e.g. trypsin, chymotrypsin). If, however, the enzymic properties in the presence of solvents are considered, a striking similarity can be found to some intracellular enzymes (e.g. aldolase, D-glyceraldehyde3-phosphate dehydrogenase3,4). The author is indebted to Professor F. B. STRAUB for valuable discussion and to Mrs. E. T6TH for the excellent technical assistance.

Institute of Biochemistry, Hungarian Academy of Sciences, Budapest (Hungary)

PAL E L O D I

1 j. T. YANG AND P. DOTY, J. Am. Chem. Soc., 79 (1957) 761. 2 R. E. \VEBB AND C. H. TANFORD, J. Am. Chem. Soc., 81 (1959) 3255. 3 p. EL6DI, Biochim. Biophys. Aeta, 44 (196o) 61o. 4 p. EL6DI, Acta Physiol. Acad. Sci., Hung., in the press. 5 M. R. McDONALD in S. P. COLOWlCK AND N. O. KAPLAN, Methods o[ Enzymology, Vol. 2, Academic Press, Inc., New York, I955, p. 887. 6 G. WOODWARD, J . Biol. Chem., 156 (1944) 143. 7 D, FINDLAY, A. P. MATHIAS AND B. R. RABIN, Nature, I87 (196o) 6Ol.

Received May 3oth, 1961 Biocl~im. Biophys. Acta, 53 (t901) 218-219