Reactivity of the tryptophan residues in bovine pancreatic deoxyribonuclease with N-bromosuccinimide.

DNase, containing 3 tryptophan residues, can be inactivated by modification of this amino acid with Nbromosuccinimide. Chromatography of acid hydrolysates shows all three tryptophan residues modified by the time inactivation is complete. However, spectrophotometric measurement indicates that only 2 residues are modified. The discrepancy is due to the unreliability of the spectrophotometric method because the absorbance decrease at 280 nm changes with time. Examination of the inactivation kinetics by the hydrolytic method reveals that 1 of the 3 tryptophan residues is crucial for enzymic activity; the other 2 residues are less important. The sequence of modification of the 3 tryptophans is affected by pH. At pH 4.0, the 2nd residue modified is most crucial; at pH 5.5, the 3rd residue modified is most crucial. Assignment of the three positions of modification in relation to their importance in catalytic activity is accomplished as follows. (a) Hydrolysis of DNase with a mixture of chymotrypsin and trypsin yields Trp-155 as the free amino acid. (6) Hydrolysis of DNase with chymotrypsin yields three tryptophan-containing peptides which are resolved by gel filtration chromatography on Sephadex G-25. The results from these analyses show that Trp178 is most reactive with N-bromosuccinimide and is not a crucial residue; Trp-155 is most crucial for enzymic activity whether it reacts more or less rapidly (at pH 4 and 5.5, respectively) than the 3rd, nonessential residue, “191. Polyacrylamide gel electrophoresis of modified DNase in sodium dodecyl sulfate shows peptide bond cleavage at oxidized tryptophan residues. Analysis of the gel pattern suggests that cleavage of peptide bonds is incomplete and that the extent of cleavage varies significantly among the 3 susceptible residues. Inactivation is directly related to the oxidation step, rather than the cleavage step, since approximately 40% of a completely inactivated sample shows no peptide bond cleavage.