Abstract
Abstract Bovine pancreatic deoxyribonuclease contains 3 tryptophan residues per molecule of enzyme (mol wt 31,000). Two of these residues react with the chemical modifying reagents specific for tryptophan, N-bromosuccinimide (NBS) and 2-hydroxy-5-nitrobenzyl bromide (HNB). A 6-fold excess of NBS over DNase completely inactivates DNase and at 100% loss of activity, one tryptophan is oxidized to oxindole per DNase molecule. HNB also reacts with a single tryptophan residue in DNase to give a 20% to 30% loss of activity. HNB-modified DNase then can be inactivated with a 6-fold excess of NBS with the resulting oxidation of a single tryptophan residue. This indicates that there are two reactive tryptophans in DNase, one of which is essential for catalytic activity. NBS also reacts with tyrosine, histidine, and methionine, but amino acid analysis of NBS-inactivated DNase showed that no other amino acids except tryptophan were modified. The structure of the NBS-inactivated enzyme has not been grossly altered. Calcium protects one of the two disulfide bonds in native DNase from mercaptoethanol reduction and also protects one disulfide bond in the inactivated enzyme. Comparison of circular dichroism spectra of NBS-inactivated and native DNase shows little significant difference. However, calcium was found to induce an ultraviolet difference spectrum in DNase while the NBS-inactivated DNase showed no difference spectrum over a wide range of calcium concentration.
Highlights
ZHydroxy5nitrobenzyl bromide purchased from Sigma was used without further purification
The percentage of initial activity is proportional to the number of tryptophans oxidized (Fig. 2) and suggests that the inactivation of DNase is caused by the destruction of a single tryptophan
The same experiment repeated on a sample of DNase purified from a different commercial batch gave 1.13 tryptophan residues oxidized at 100% loss of activity
Summary
MateriaZsDNase, DP grade, and calf thymus DNA were purchased from Worthington. ZHydroxy5nitrobenzyl bromide purchased from Sigma was used without further purification. Preparation of DBase-Worthington DP grade DNase was purified over sulfoethyl-Sephadex by a modification of the procedure described earlier [12] in which CaCh. at a concentration of. 5 mM was included in all elution buffers. DNase from inactivation by contaminating proteolytic enzymes the earlier procedure of diisopropyl fluorophosphate treatment prior to chromatography of DNase proved to be unnecessary. The elution profile obtained by the modified procedure was identical with that described earlier [12]. The DNase A fraction obtained from sulfoethyl-Sephadex chromatography was concentrated by ultrafiltration, dialyzed against 0.25 M acetate buffer at pH 4.70, and chromatographed over a phosphocellulose column (2 x 100 cm) as described by Salnikow, Stein, and
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