Effect of controlled redox potential and dissolved oxygen on the in vitro refolding of an E. coli alkaline phosphatase and chicken lysozyme.

The development of efficient purification strategies of recombinant active protein derived from inclusion bodies requires the knowledge of the effect of environmental variables, such as redox potential (RP) and dissolved oxygen tension (DOT), in order to control the protein folding process. However, that information is scarce and only few in vitro studies of the impact of such variables have been reported under constant controlled conditions. In this work, the effect of controlled RP and DOT on the refolding of E. coli alkaline phosphatase (AP) and chicken lysozyme (CL) enzymes were studied. Disulphide bonds of both enzymes were reduced in an instrumented vessel using 2-mercaptoethanol and nitrogen. In the latter case, guanidine hydrochloride was also used to denature the protein. Such conditions caused protein conformational changes, as determined by the intrinsic fluorescence spectra that correlated with a decrease on the activity in both cases. Reduced enzymes were then oxidized, under different constant and predetermined RP or DOT, by manipulating the gas composition in the vessel. Folding kinetics were followed as the recovery of enzyme activity. Results showed that the percentage of recovery and rate of increase of enzymatic activity directly depended on the RP and DOT. A higher folding efficiency was found under controlled DOT compared to controlled RP conditions. These results are useful for establishing protein folding strategies to improve the recovery of active protein from inclusion bodies.

Complex, unusual conformational changes in kidney betaine aldehyde dehydrogenase suggested by chemical modification with disulfiram.

The NAD+-dependent animal betaine aldehyde dehydrogenases participate in the biosynthesis of glycine betaine and carnitine, as well as in polyamines catabolism. We studied the kinetics of inactivation of the porcine kidney enzyme (pkBADH) by the drug disulfiram, a thiol-reagent, with the double aim of exploring the enzyme dynamics and investigating whether it could be an in vivo target of disulfiram. Both inactivation by disulfiram and reactivation by reductants were biphasic processes with equal limiting amplitudes. Under certain conditions half of the enzyme activity became resistant to disulfiram inactivation. NAD+ protected almost 100% at 10 microM but only 50% at 5mM, and vice versa if the enzyme was pre-incubated with NAD+ before the chemical modification. NADH, betaine aldehyde, and glycine betaine also afforded greater protection after pre-incubation with the enzyme than without pre-incubation. Together, these findings suggest two kinds of active sites in this seemingly homotetrameric enzyme, and complex, unusual ligand-induced conformational changes. In addition, they indicate that, in vivo, pkBADH is most likely protected against disulfiram inactivation.