Fluorometric study of the acid-induced denaturation of Staphylococcal nuclease and its mutant forms.

Abstract

The acid-induced denaturation of wild-type Staphylococcal nuclease (WT) and its eight mutant forms, L25A, V66L, G79S, A90S, G88V, H124L, V66L/G88V, and V66L/G79S/G88V, was investigated using Trp140 fluorescence as a probe at 30 degrees C. The values of pH(1/2), at which the denaturation is half completed, and n, the apparent number of protons which trigger the denaturation and are taken up by the proteins upon denaturation at pH(1/2), were evaluated from the pH dependence of the fluorescence intensity. The values of pH(1/2) and n for WT were 3.8 and 1.8 respectively. The amino acid replacements changed the pH(1/2) values to a range between 3.0 (H124L) and 4.4 (G79S) and also changed the n values to a range between 1.0 (A90S) and 3.0 (G88V). There was a negative correlation between the values of pH(1/2) and n. It was suggested that the amino acid replacements may change the energy levels of the native state and/or the denatured state mainly in the neutral (stable) pH region, not in the acidic (unstable) region, resulting in the correlative changes in pH(1/2) and n.