A Kinetic Study of the Complementation of Fragments of Staphylococcal Nuclease

Autor: Hiroshi Taniuchi, Raymond F. Chen, Albert Light
Rok vydání: 1974
Předmět:
Zdroj: Journal of Biological Chemistry. 249:2285-2293
ISSN: 0021-9258
Popis: The complementation of fragments of staphylococcal nuclease to form an ordered structure was studied by stopped flow kinetic measurements. The increase in the intensity of tryptophan fluorescence of residue 140 on complementation was followed. The complementation of two fragments containing residues 6 to 48 and residues 49 to 149 (or 50 to 149) to form Nuclease-T' follows apparent first order kinetics, both with regard to variation of time and fragment concentration accessible to study. The first order rate constant of the formation of Nuclease-T' is in the range of 0.03 to 0.05 s-1, which is much less than that of refolding of acid-denatured intact nuclease observed by Schechter et al. (Schechter, A. N., Chen, R.F., and Anfinsen, C.B. (1970) Science 167, 886–887). The change of temperature from 5° to 45° caused only a small increase in the rate of formation of Nuclease-T'. The presence of the ligands, thymidine 3',5'-diphosphate and Ca2+, and the change of pH of the reaction mixture from 5 to 8 had no effect on the rate constant. The complementation between the fragments of residues 1 to 126 and of residues 99 to 149 and between the fragments of residues 1 to 126 and of residues 49 to 149 (or 50 to 149) also fits first order kinetics and shows rate constants equivalent to that of Nuclease-T'. Examination of the kinetic equation for the simplest model, in which specific prefolding of each fragment is required for the productive collision, and the collision is the rate-limiting step of the complementation, fails to explain these results. However, the results may be interpreted by assuming the rate-limiting step either to be the prefolding of one of the two fragments, which then combines with the other fragment, or the folding of a disordered intermediate complex formed by the two fragments.
Databáze: OpenAIRE