Rate Of Refolding Research Articles

Antibody as an immunological probe for studying the refolding of bovine serum albumin. I. The catalysis of reoxidation of reduced bovine serum albumin by glutathione and a disulfide interchange enzyme.

We have used an immunochemical approach to study the refolding of bovine serum albumin. Using antibody as a probe for return of native structure, we have been able to demonstrate the regeneration of native structure at several sites on the surface of the molecule. Using this technique, we have shown that the rate of refolding of reduced bovine serum albumin catalyzed by either glutathione or rat liver disulfide interchange enzyme is greater than the rate of air reoxidation of albumin. The half-regeneration times for albumin, however, are substantially greater than those obtained with smaller proteins that have fewer disulfide bonds. We have also demonstrated that the reoxidized monomers isolated at the end of the refolding process are immunologically identical to native monomers. In addition, the tryptophan fluorescence emission maxima were the same as that of the native monomers.

A quantitative treatment of the kinetics of the folding transition of ribonuclease A.

New experimental data and a quantitative theoretical treatment are given for the kinetics of the thermal folding transition of ribonuclease A at pH 3.0. A three-species mechanism is used as a starting point for the analysis: U1 (slow) in equilibrium U2(fast) in equilibrium N, where U1 and U2 are two forms of the unfolded enzyme with markedly different rates of refolding and N is the native enzyme. This mechanism is based on certain facts established in previous studies of refolding. The kinetics of unfolding and refolding show two phases a fast phase and a slow phase, over a range of temperatures extending above the transition midpoint, Tm. The three-species mechanism can be used in this range. At higher temperatures a new much faster kinetic phase is also observed corresponding to the transient formation of a new intermediate (I). Although the general solution for a four-species mechanism is complex it is not difficult to extend the three-species analysis for the special case found here, in which the fast reaction (I in equilibrium N) is well separated from the other two reactions. At temperatures below the transition zone the slow phase of refolding becomes kinetically complex. No attempt has been made to extend the analysis to include this effect. The basic test of the three-state analysis is the prediction as a function of temperature of alpha2, the relative amplitude of the fast phase, both for unfolding and refolding. At temperatures above Tm for which the three-state analysis must be extended to include the new intermediate I, a crresponding quanitity alpha2(cor) is predicted and compared with measured values. Data used in the three-state prediction are values of tau2 and tau1, the time constants of the fast and slow kinetic phases, plus a single value of alpha2 measured when tau2 and tau1 are well separated. The observed and predicted values of alpha2 agree within experimental error. The analysis predicts correctly that, for these experiments, alpha2 should have the same value in unfolding as in refolding in the final conditions. The analysis also predicts satisfactorily the equilibrium transition curve from kinetic data alone. Four striking properties of the kinetics are explained or correlated by the analysis: (a) the drop in alpha2 to a minimum near Tm as well as the delayed rise in alpha2 above Tm;(b) the vanishing of alpha1 above the transition zone; (c) the sharp drop in tau1 inside the transition zone followed by a partial leveling off outside this zone; and (d) the passage of tau2 through a maximum near Tm. Through a comparison of observed and predicted values of alpha2, the analysis also rules out the alternative three-species mechanism U1 (slow) in equilibrium N (fast) in equilibrium U2. Finally, the temperature dependence of the amplitude for the fast reaction (I in equilibrium N) is discussed; the behavior of I is like that of U2 and I may be an unfolded species populated at equilibrium...

A physical difference between the fast- and slow-refolding forms of nitrotyrosyl ribonuclease A: the p K values of the nitrotyrosyl groups

In the preceding paper we present kinetic evidence for a slow equilibrium between two conformational forms of heat-unfolded ribonuclease A whose rates of refolding differ 100-fold. In a search for physical differences between these two forms, we undertook a study of the p K changes during refolding of a specific set of freely ionizing surface groups. By use of a standard procedure the three freely ionizing tyrosine groups (p K ∼- 10) have been nitrated by tetranitromethane, yielding three nitrotyrosine groups (p K ∼- 6.8). Nitrotyrosyl ribonuclease A closely resembles the unmodified enzyme as regards: (1) enzymatic activity; (2) thermal unfolding transition at neutral pH; and (3) kinetics of refolding. In particular, stopped-flow measurements of 2′CMP binding during refolding show that the fast-refolding reaction is unchanged by nitration and yields fully folded enzyme able to bind 2′CMP. The p K change of the nitrotyrosyl groups upon refolding is quite different in the fast- and slow-refolding reactions. In the slow reaction it is small (− 0.046 ± 0.006 pH unit) but easily measureable, whereas in the fast-reaction it is too small to be detected (− Δp K less than 0.02 pH unit). This difference in p K change upon refolding can be attributed to different p K values of the nitrotyrosyl groups in the slow-refolding and fast-refolding forms of the heat-unfolded protein. Presumably the same structural differences between these two heat-unfolded forms are responsible both for the p K difference and for the 100-fold difference in rates of refolding. These results support the simple three-species mechanism for refolding discussed in the preceding paper. (a) They demonstrate a physical difference between the fast- and slow-refolding species. (b) They do not show any additional kinetic complexity when refolding is measured by a property that distinguishes between the fast- and slow-refolding species.

Both the Fast and Slow Refolding Reactions of Ribonuclease A Yield Native Enzyme

The fast reaction ((T2) approximately 50 msec) observed previously in the refolding of thermally unfolded ribonuclease A (disulfide bonds intact) has now been studied by two properties indicative of enzyme function: binding of a competitive inhibitor (2'CMP) and hydrolysis of a substrate (CpA --> C > p + A). Both the binding and catalytic reactions are fast (<2 msec) compared to refolding. Binding of 2'CMP occurs during both fast and slow refolding reactions, and the protein folded in the fast reaction has a normal binding constant for 2'CMP. Recovery of enzymatic activity during the fast refolding reaction, as measured by the rate of CpA hydrolysis, parallels the kinetic curve for 2'CMP binding. When the kinetics of refolding are measured by the burying of exposed tyrosine groups, no difference is found. The presence of 2'CMP has no effect on the kinetics of refolding. The results show that the fast refolding reaction does not yield an intermediate in the refolding of RNase A. Instead, both fast and slow refolding reactions have a common product, fully active RNase A. Although they show a 100-fold difference in rates of refolding, the starting materials for the fast and slow refolding reactions have similar properties, as regards: (a) the molar absorbancy at 286 nm, reflecting the state of exposed tyrosine groups, and (b) their apparent failure to bind 2'CMP.

Some catalytic and structural aspects of reactivation of denatured citrate synthase

Crystalline pig heart citrate synthase, denatured in 6.0 M guanidine hydrochloride, was renatured in vitro by dilution. The initial presence of substrate or high ionic strength increased the rate and final extent of refolding. The rate of reactivation was inversely related to concentration of denatured citrate synthase. Citrate synthase undergoes reversible structural transitions as measured by changes in tryptophan fluorescence.