Paramagnetic and Fluorescent Probes Attached to “Buried” Sulfhydryl Groups in Human Carbonic Anhydrases. Application to Inhibitor Binding, Denaturation and Refolding

Abstract

1. The single -SH groups in the human carbonic anhydrases B and C have been modified under denaturing conditions. The modified enzymes recover catalytic activity after dilution of the denaturing medium with buffer. By this method a spin label and a fluorescent probe were specifically introduced into the molecules. 2. The modified and reactivated enzymes have similar kinetic properties, inhibitor-binding constants, circular dichroism spectra, and stabilities towards guanidine hydrochloride as the native enzymes. However, the esterase activity of the modified C enzyme is reduced to about 50%. 3. The spectra associated with the probes are insensitive to inhibitor binding in case of the B enzyme, whereas changes of electron paramagnetic resonance spectrum and fluorescence intensity respectively, were observed for the probe-containing C enzymes. The cysteines are located in different parts of the tertiary structures of the homologous B and C enzymes, and these observations suggest that small conformational changes accompanying inhibitor binding are localized to regions of the molecules near the active-site cavity. 4. During denaturation of the spin-labeled B enzyme in 1.7 M guanidine hydrochloride a transient mobilization of the probe occurs, but the mobility is ultimately reduced to a low level. This observation supports previous evidence that denaturation under these conditions, in or near the transition region, mainly yields incorrectly folded molecules rather than stable intermediates between native and fully denatured molecules. 5. During refolding of fully denatured, spin-labeled B and C enzymes the mobility of the probe is drastically reduced within less than 0.1 s after dilution. This would reflect a very short lifetime of the randomly coiled state under these conditions.