Alkylation of an Essential Histidine Residue in Porcine Spleen Deoxyribonuclease

Preparations of porcine spleen deoxyribonuclease (DNase II) obtained by the ammonium sulfate precipitation procedure of Bernardi et al. (Bernardi, G., Bernardi, A., and Chersi, A. (1966) Biochim. Biophys. Acta 129, 1) can be purified by sequential chromatography over sulfoethyl-Sephadex and phosphocellulose at pH 4.6. The purified enzyme is homogeneous by the criteria of chromatography on carboxymethyl-Sephadex, gel electrophoresis at pH 3.8, and gel filtration on Sephadex G-100. The specific activity, molecular weight, and amino acid composition of the purified protein are close to the values obtained by Bernardi et al. (Bernardi, G., Apella, E., and Zito, R. (1965) Biochemistry 4, 1725) on DNase II purified by a different chromatographic procedure. Significant differences between the two protein preparations are found only in tryptophan content and extinction coefficient at 280 nm. DNase II can be inactivated completely by iodoacetate at pH 4.6. The neutral alkylating agent, iodoacetamide, is without effect. At 2 x 10-5 m iodoacetate, only 4 min are required for half-inactivation. This inactivation rate is sufficiently rapid to permit the titration of DNase II activity with molar equivalent of iodoacetate. Loss of enzyme activity with radioactively labeled iodoacetate parallels the incorporation of 1 carboxymethyl group per molecule. The inactivated protein can be separated from active enzyme by chromatography over phosphocellulose. Inactive enzyme obtained by chromatography has 1 residue of 3-carboxymethylhistidine as the sole product of iodoacetate reaction. This alkylation reaction probably occurs in the active site region, since the enzyme is protected completely from inactivation by iodoacetate when substrate DNA is present at a concentration of 0.5 mg per ml.