SPIN-LABELING STUDY OF HUMAN SERUM-ALBUMIN IN REVERSE MICELLES

Human serum albumin (HSA), spin labeled at the sulfhydryl group by the reagent 3-maleimidoproxyl (3MAL), was studied in reverse micelles formed by sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The electron spin resonance spectra were recorded at different water contents and analyzed by computer simulations. In order to obtain agreement between experimental and calculated spectra, the use of an anisotropic model of reorientational diffusion for 3MAL-HSA in reverse micelles was necessary. An isotropic reorientational motion was suitable to simulate the 3MAL-HSA spectrum in aqueous solution. This result suggests that conformational changes of the protein which modify the label environment occur in reverse micelles. The rotational correlation times for 3MAL-HSA in reverse micelles were strongly dependent on the water content: the protein experienced a more hindered environment for rotational diffusion as the water pool size decreased.