Ruiz Silva B E, Koepf E K, Burtnick L D, Turro N J
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Biochem Cell Biol. 1992 Jul;70(7):573-8. doi: 10.1139/o92-088.
Horse plasma gelsolin was labelled with the sulfhydryl-specific fluorescent reagent N-(1-pyrenyl)iodoacetamide. The level of incorporation of probe was 1.6 +/- 0.3 mol pyrene/mol gelsolin. The circular dichroism spectrum of pyrenyl-gelsolin and its ability to interact with muscle actin were not different from that found for unmodified gelsolin. The emission from pyrenyl-gelsolin was dominated by a broad emission band centred near 483 nm, characteristic of the presence of pyrene excimers. Analysis of excitation spectra for the monomer and excimer-type fluorescence suggested that ground-state interactions may occur between adjacent pyrenes in the gelsolin structure. In the case either of excimer formation or of ground-state pyrene-pyrene interactions in doubly labelled gelsolin molecules, the modified Cys residues must be in close proximity in the folded protein structure. Thermal denaturation of gelsolin could be monitored by observing the decrease in excimer emission that accompanied heating and unfolding of the tertiary structure. While heat treatment alone did not eliminate excimer fluorescence, digestion of gelsolin with chymotrypsin completely abolished such emission. Also, pyrenyl-gelsolin prepared and studied in 6 M guanidine-HCl exhibited fluorescence characteristic of pyrene monomers exclusively.
马血浆凝溶胶蛋白用巯基特异性荧光试剂N-(1-芘基)碘乙酰胺进行标记。探针的掺入水平为1.6±0.3摩尔芘/摩尔凝溶胶蛋白。芘基-凝溶胶蛋白的圆二色光谱及其与肌肉肌动蛋白相互作用的能力与未修饰的凝溶胶蛋白无异。芘基-凝溶胶蛋白的发射以位于483nm附近的宽发射带为主，这是芘激基缔合物存在的特征。对单体和激基缔合物型荧光的激发光谱分析表明，凝溶胶蛋白结构中相邻芘之间可能发生基态相互作用。在双标记凝溶胶蛋白分子中激基缔合物形成或芘-芘基态相互作用的情况下，修饰的半胱氨酸残基在折叠的蛋白质结构中必须紧密相邻。凝溶胶蛋白的热变性可以通过观察伴随三级结构加热和展开的激基缔合物发射的减少来监测。虽然单独的热处理并没有消除激基缔合物荧光，但用胰凝乳蛋白酶消化凝溶胶蛋白完全消除了这种发射。此外，在6M盐酸胍中制备和研究的芘基-凝溶胶蛋白仅表现出芘单体的荧光特征。
