Fluorescence of equine platelet tropomyosin labeled with acrylodan.
Equine platelet tropomyosin was labeled with the sulfhydryl-specific fluorescent reagent 6-acryloyl-2-dimethylaminonaphthalene (acrylodan). The extent of labeling at 4 degrees C could be regulated between 0.5 and 1.3 acrylodans per tropomyosin chain by varying the reaction time from 1 to 4.5 h. Acrylodan-labeled platelet tropomyosin, AD-P-TM, was highly fluorescent, having an emission maximum near 518 nm on excitation at 365 nm. Steady-state measurements of polarization of the fluorescence of AD-P-TM in both low and high ionic strength solutions gave Perrin plots that exhibited sharp changes in slope near 50 degrees C, indicative of a sharp increase in mobility of the label at that temperature. This correlates with the melting temperature of the platelet tropomyosin coiled coil observed by circular dichroism [G. P. Côté, W. G. Lewis, M. D. Pato, and L. B. Smillie, (1978) FEBS Lett. 91, 237-241]. Perrin plots of carboxypeptidase A-treated platelet tropomyosin that was labeled with acrylodan after digestion resembled more closely those of acrylodan-labeled cardiac tropomyosin rather than those of AD-P-TM, suggesting that the observed emission arose from label at Cys-153 on each truncated platelet tropomyosin chain. In solutions containing 150 mM KCl and 5 mM MgCl2, addition of actin at up to a sixfold molar excess over AD-P-TM caused both the fluorescence emission intensities and fluorescence polarization values of samples to increase. In the presence of actin, the wavelength of maximal emission was shifted to shorter values by about 5 to 7 nm. These changes indicate that actin does bind to AD-P-TM and that the binding affects the environment of the label, both by making it more hydrophobic and by reducing the freedom of the label to tumble in solution.
Clark
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Department of Chemistry, University of British Columbia, Vancouver, Canada.
Burtnick
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LD
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000199:Actins / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001792:Blood Platelets / Q000032:analysis*; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006736:Horses / Q000097:blood*; D006358:Hot Temperature; D009281:Naphthalenes; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000097:blood*; D014357:Trypsin
D000199:Actins; D000596:Amino Acids; D005456:Fluorescent Dyes; D009281:Naphthalenes; D014335:Tropomyosin; C038518:acrylodan; D015081:2-Naphthylamine; D014357:Trypsin