A reversibly refolding non-covalent complex of three peptides from Staphylococcus aureus penicillinase [proceedings

Abstract

Reduced and ‘scrambled’derivatives of NO2-ribnuclease appear to be less stable than the corresponding derivatives of unmodified ribonuclease. In our hands, reduced ribonuclease is stable in solutions of pH4 for 4-5 days at 4”C, whereas reduced NO,-ribonuclease is reoxidized with a half-time of several hours. Likewise, the background enzymic activity of ‘scrambled‘ NO,-ribonuclease increases during storage in solution at pH8.0, 4“C, whereas scrambled ribonuclease is comparatively stable. Although this instability may be a limitation, the nitrotyrosine residue in these derivatives is a useful probe of the conformational changes accompanying disulphide-formation and interchange. The conversion from ‘scrambled‘ to ‘native’ NO,-ribonuclease corresponds to a vertical transition between the titration curves of Fig. 1. As a result of the shift in PIC., if this conversion is carried out between pH6.0 and 7.4, it should be accompanied by a significant change in A428. We find that this conversion is catalysed by protein disulphide isomerase (EC 5.3.4.1), an enzyme catalysing thiol-disulphide interchange, and hence disulphide-bond isomerization in proteins (Freedman & Hawkins, 1977). Incubation of ‘scrambled‘ NO,-ribonuclease with partially purified protein disulphide isomerase (Hawkins & Freedman, 1976) in the standard conditions for assay of this enzyme (Ibbetson & Freedman, 1976) leads to the appearance of increased ribonuclease activity and an increase in A42s. The rate of increase in ribonuclease activity with ‘scrambled’ NO,-ribonuclease is 2-10 % that found with scrambled unmodified r i b nuclease. With ‘scrambled’ NO,-ribonuclease in the range O.l-O.Smg/ml, rates of increase in absorbance were 0.0035-0.0125 AA428 units/min per mg of enzyme. So nitrated ribonculease derivatives may provide useful probes for the kinetic characterization of disulphide-bond formation and isomerization processes, including those catalysed by protein disulphide isomerase.