Fluorescent responses of acrylodan-labeled plasma gelsolin.
Under nondenaturing conditions, 1 mol of horse plasma gelsolin reacts with 1.9 +/- 0.5 mol (mean +/- SD, n = 6) of the sulfhydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The degree of labeling in 6 M guanidine-HCl increases to about 3 mol of acrylodan per mole of gelsolin. Viscosity studies show that the modified gelsolin retains its ability to sever F-actin filaments. Circular dichroism spectra in the peptide bond absorption region are indistinguishable for labeled and unmodified gelsolin at room temperature. The thermal stability of gelsolin, as monitored by circular dichroism, is unaffected by reaction with acrylodan. While circular dichroism spectra of acrylodan-labeled gelsolin recorded at room temperature are not influenced significantly by Ca2+, fluorescence studies reveal a number of Ca(2+)-dependent changes in the protein. Ca2+ causes a decrease and red-shift in fluorescence emission, an increase in sensitivity to quenching by I-, and a decrease in polarization of the fluorescence of acrylodan-labeled gelsolin. Together, these changes in fluorescence properties indicate there to be an increased exposure of the label to the solvent when gelsolin binds Ca2+. Fluorescence polarization experiments in which acrylodan-labeled gelsolin is titrated with actin emphasize that Ca2+ is required for these two proteins to interact.
Reid
S W
SW
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Koepf
E K
EK
Burtnick
L D
LD
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000097:blood* / Q000737:chemistry; D002942:Circular Dichroism; D004355:Drug Stability; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D018260:Gelsolin; D019791:Guanidine; D006146:Guanidines; D006736:Horses; D006358:Hot Temperature; D008840:Microfilament Proteins / Q000097:blood* / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014783:Viscosity
D000199:Actins; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D018260:Gelsolin; D006146:Guanidines; D008840:Microfilament Proteins; D004533:Egtazic Acid; C038518:acrylodan; D015081:2-Naphthylamine; D019791:Guanidine; D002118:Calcium