Comparison of the dynamic structure of alpha-chymotrypsin in aqueous solution and in reversed micelles by fluorescent active-site probing.
A highly fluorescent anthraniloyl (Ant) group was covalently attached to the active site of alpha-chymotrypsin (CT), probably at Ser195. Ant-CT is stable at neutral pH for months, allowing a detailed fluorescence study of Ant-CT as a model protein to investigate its physical properties in 0.1 M Tris/HCl, pH 8.2, and in reversed micelles of n-octane, 0.1 M Tris/HCl, pH 8.2, and sodium bis(2-ethylhexyl)sulfosuccinate (AOT). Steady-state fluorescence measurements of the progressive red-shift of the center of gravity of the emission band as function of degree of hydration, wo, defined as [H2O]/[AOT], indicate that the average polarity in the vicinity of the probe is approaching that of bulk water at wo > 12. Time-resolved fluorescence measurements of Ant-CT in water and in reversed micelles showed that the active site has different properties in reversed micelles compared to those in water. Some specific changes at very low water content (0.6 < wo < 5) can be observed, which correlate with enzyme activity measurements in the same wo region (unpublished results). These effects are, for instance, significant changes in the average fluorescence lifetime and the internal flexibility of the probe. The overall rotational-correlation time of the enzyme in AOT reversed micelles seems to be independent on wo (5 < wo < 29), which suggests that the enzyme creates its own micelle.
Dorovska-Taran
V N
VN
Department of Biochemistry, Agricultural University Wageningen, The Netherlands.
Veeger
C
C
Visser
A J
AJ
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008823:Micelles; D009038:Motion; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014867:Water
D005456:Fluorescent Dyes; D008823:Micelles; D012996:Solutions; D014867:Water; D002918:Chymotrypsin