Reversible thermal denaturation of immobilized rhodanese.

摘要: For the first time, enzyme rhodanese had been refolded after thermal denaturation. This was previously not possible because of strong tendency for soluble to aggregate at temperatures above 37 degrees C. The present work used that covalently coupled a solid support under conditions were found preserve activity. Rhodanese immobilized using an N-hydroxymalonimidyl derivative Sepharose containing 6-carbon spacer. number competent active sites measured by [35S]SO3(2-) form site persulfide is obligatory catalytic intermediate. Soluble irreversibly inactivated in 10 min 52 regained least 30% its original activity even boiling 20 min. Km and Vmax each approximately 3 times higher than corresponding values native enzyme. After preincubation high temperatures, progress curves showed induction periods up 5 before attaining apparently linear steady states. pH dependence same both These results indicate significant stabilization immobilization, instabilities caused adventitious solution components are sole reasons irreversibility denaturation seen with consistent models invoke protein association as major cause inactivation Furthermore, period studies which show refolding proceeds through intermediate