680 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 36095

INACTIVATION OF PANCREATIC RIBONUCLEASE WITH

HYDROXYLAMINE-OXYGEN-CUPRIC ION

Y-C. L I N

National Taiwan University, Chemistry Research Centre, Roosevelt Road Section 4, Taipei, Taiwan (China)

(Received December I3th, 1971)

SUMMARY

In the studies of ribonuclease, it was found that a system containing hydroxyl- amine, Cu ~+ and 0 2 will inactivate its biological activity. All three components are es- sential for the inactivation. The inactivation of ribonuclease by this system is pH- dependent. The rate of inactivation was studied as a function of hydroxylamine con- centration and Cu 2+ concentration. It is interesting to note that both Tris and phos- phate serve as competitive inhibitors. It was also found that all four histidine residues of ribonuclease were missing when the inactivated ribonuclease was subiected to acid hydrolysis followed by amino acid analysis.

INTRODUCTION

Hydroxylamine has been used extensively in both organic chemistry and bio- chemistry, and its general properties as a nucleophile have been thoroughly studied and are well understood. However, several reports in the qiterature on the inhibition of enzymes such as urease 1, alcohol dehydrogenases 2, xanthosine-5'-phosphate amin- ase a and carboxylesterasO by hydroxylamine suggest that the reagent also may par- ticipate in reactions which apparently are not so well understood. I t has been sug- gested that hydroxylamine may act as an oxidizing agent 5, and a recent report has demonstrated that hydroxylamine together with Cu 2+ and oxygen is an effective hydroxylating reagenO.

In the course of our studies on pancreatic ribonuclease, we found that this en- zyme was rapidly inactivated in the presence of hydroxylamine. We also found that Cu 2+ and oxygen were obligatory participants in the inactivation reaction, but in contrast to the previous report where hydroxylation on tyrosine appeared to be the primary reaction 4, the loss of ribonuclease activity was found to be associated with destruction of histidine. It is the purpose of this report to document this finding and to suggest that the modification of proteins by the combination of hydroxylamine, Cu "+ and oxygen may be quite general and could involve several different amino acid residues.

Biochim. Biophys. Acta, 263 (1972) 68o-682

INACTIVATION OF RIBONUCLEASE 681

When bovine pancreatic ribonuclease A (5 × recrystallized, Sigma Chemical Co.) was incubated at room temperature with o.I M hydroxylamine, l O -4 M Cu 2+ at pH 7.5, and stirred by gently flushing the solution with a fine stream of oxygen through a capillary tubing, the activity (determined by the method of Crook et al. 6) was rapidly lost (Fig. ia). I f any of the three components, hydroxylamine, Cu 2+ or 02, were omitted, no inactivation was observed. I t is also interesting to note that the inactiva- tion was faster in phosphate buffer than in Tris buffer. The effect of hydroxylamine concentration on the rate of inactivation is given in Fig. Ib, and the effect of Cu 2+ in Fig. ic. While the system shows normal saturation levels for Cu 2+ at a fixed level of hydroxylamine and 02, a clear optimum in the hydroxylamine concentration is ob- served at constant Cu 2+ and 02. Both of these features must be accounted for when the mechanism of the reaction is elucidated. The pH profile for the inactivation (Fig. Id) suggests that a group with a pH of about 5.2 is involved; this provided the first

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Fig. I. (a) The inactivation of ribonuclease (i mg/ml) by o.i M hydroxylaminc Io -4 M Cu 2+ in a solution cont inuously sa tura ted with oxygen in o.i M phosphate buffer, p H 7.5 ( C ) - - C ) ) and in o.I M Tris buffer, p H 7.5 ( O - - - Q). A /X, results wi th an unt rea ted control sample, and the results obtained when N 2 gas was subs t i tu ted for oxygen, when hydroxy lamine was omit ted or when Cu 2+ was omit ted and E D T A was added to remove traces of Cu ~+ presen t in the water. (b) The effect of hydroxylamine concentrat ion on the rate of inactivation of r ibonuclease in the presence of io -~ M Cu ~+ and in 0.2 M phospha te buffer, p H 7.0, cont inuously sa tura ted with oxygen. The same profile was obtained wi th Tris buffer, bu t with less inactivation. Percent in- act ivat ion represents the loss of act ivi ty after i min incubat ion relative to the zero t ime value for each sample. (c) The effect of Cu ~+ concentrat ion on the rate of inactivation of r ibonuclease in the presence of o.i M hydroxylamine and in 0.2 M phospha te buffer, p H 7.0, cont inuously sa tura ted with oxygen. Inact iva t ion was evaluated as in (b). (d) The effect of p H on the ra te of inact ivat ion of ribonuclease in the presence of Io -3 M Cu ~+, o.I M hydroxylamine in solutions cont inuously sa tura ted with oxygen.

indication that one of the active site histidines 7 could be involved in the inactivation process. This was confirmed by amino acid analysis. A comparison of the amino acid composition of native and inactivated enzyme, showed only one significant and con- sistent difference, namely the absence of 3.5-4.0 histidine residues in the inactivated

Biochim. Biophys. Acta, 263 (1972) 68o-682

682 Y-C. LIN

enzyme (of a total of 4 in the native enzyme). We were unable to demonstrate any specificity in the reaction which appears to modify all 4 histidine residues in a totally or very nearly totally random fashion. Minor changes in the level of other amino acids were sometimes observed after inactivation, notably in cysteine, tyrosine and phenylalanine, but these were too small to account for the complete inactivation of the enzyme. Another significant feature of the amino acid analyses is the fact that the hydrolysate of the inactivated enzyme showed a consistent and significant increase in aspartic acid and glycine and that small amounts of urea also were present. From subsequent studies on model reactions it has been found that hydrolysis of N-acetyl- histidine treated with hydroxylamine, Cu 2+ and 02 gives rise to these three com- pounds 8. This finding must also be accounted for in the elucidation of the reaction mechanism 8.

The main difference in our results relative to reports in the literature on the in- activation of enzymes by hydroxylamine is in the rate of the reaction. While the ribonuclease inactivation is completed in a few minutes, other enzymes apparently had to be incubated for several hours or even days before significant changes were ob- served. Since the concentration of all three reagents is important for the optimal rate, this difference should perhaps not be surprising. We have, however, tested several enzymes under our conditions and have found that the reaction is uniquely fast with ribonuclease. Egg white lysozyme, bovine chymotrypsin, rabbit muscle aldolase and pig heart fumarase were all inactivated, but the rates observed were 5 2o times slower than the rate with ribonuclease.

Attempts have been made to establish empirically which residues are modified by hydroxylamine, Cu 2+ and 0.,. Based on preliminary results with free amino acids and with N-acetylamino acids, we have found that in addition to histidine, methionine and tryptophan are rapidly modified, while tyrosine and possibly cysteine are modified more slowly. Based on this observation, it is doubtful that this process can be utilized for specific modification of proteins. However, because of the extensive use of hydro- xylamine in enzymology and protein chemistry, it is of obvious importance to be aware of these potential side reactions if traces of Cu 2+ are present.

AKNOWLEDGEMENTS

This work was supported in part by a U.S. Public Health Service grant from the National Institutes of Health (GM 15053 ). The author wishes to thank Dr Finn Wold, in whose laboratory this work was performed, for his helpful advice, many valuable discussions and critical evaluation of the manuscript.

R E F E R E N C E S

i W. N. Fishbein and P. P. Carbone, J . Biol. Chem., 24o (1965) 24o 7. 2 N. O. Kaplan and M. M. Ciotti, J. Biol. Chem., 2Ol (1953) 785 • 3 T. T. Fukuya lna and K. L. Donovan, J. Biol. Chem., 243 (1968) 5798. 4 M. T. C. Runneagar , R. L. Blakeley, R. T. Fernley, E. C. Webb and B. Zerner, Biochim.

Biophys. Acta, 167 (1968) 632. 5 D. Schuech, C. Wagenknecht , J. Wagner and P. Hykes, Acta Biol. Med. Ger., IO (1963) 459. 6 E. M. Crook, A. P. Mathias and B. R. Rabin, Biochem. J., 74 (196o) 234. 7 A. M. Crestfield, W. H. Stein and S. Moore, J. Biol. Chem., 238 (1963) 2413. 8 Y. C. Lin, J. Chinese Chem. Soc., in the press.

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