Swoboda G, Hasselbach W
Z Naturforsch C Biosci. 1985 Nov-Dec;40(11-12):863-75.
Several model compounds containing thiol and/or amino groups (mercaptoethanol, glutathione, cysteine, ethanolamine, glycine) were studied with respect to their reactivity towards fluorescein isothiocyanate (followed spectrophotometrically at 504 and 412 nm), stability of product and long-wave absorption maximum of the fluorescein residue attached. Thiol groups reacted by far more readily than amino groups. A specific effect was observed with cysteine, indicating an intramolecular transfer of the fluorescein residue from SH to NH2. With sarcoplasmic vesicles both types of reactions were observed. The ratio of products, which can be distinguished by their different stabilities and absorption spectra, depended on the absence or presence of detergents. While with native vesicles the NH2 reaction predominated, with vesicles solubilized with sodium dodecylsulfate, octaethyleneglycol mono-n-dodecyl ether or 1-0-tetradecyl-propanediol-(1,3)-3-phosphorylcholine the SH reaction became prevailing. Already 0.35 mg sodium dodecylsulfate per mg protein were sufficient to give rise to dithiourethane formation exclusively. Excess fluorescein isothiocyanate reacted with several thiol groups of dodecylsulfate-solubilized vesicles. In the presence of ATP binding of fluorescein isothiocyanate to native vesicles was significantly reduced. Total blockage of the vesicular SH groups with N-ethyl-maleimide led to preparations that reacted with fluorescein isothiocyanate much more slowly, compared to native vesicles. Octaethyleneglycol mono-n-dodecyl ether or 1-0-tetradecyl-propanediol-(1,3)-3-phosphorylcholine in the assay accelerated the thioureide formation from N-ethylmaleimide modified vesicles, whereas sodium dodecylsulfate prevented it almost completely. Our results support the suggestion that one or several thiol groups in vicinity of the highly reactive lysyl residue might play a role in the fast specific reaction, which is only observed with intact native vesicles.
研究了几种含有硫醇和/或氨基的模型化合物（巯基乙醇、谷胱甘肽、半胱氨酸、乙醇胺、甘氨酸）与异硫氰酸荧光素的反应活性（通过在504和412nm处进行分光光度法跟踪）、产物的稳定性以及连接的荧光素残基的长波吸收最大值。硫醇基团的反应远比氨基基团容易得多。观察到半胱氨酸有特殊作用，表明荧光素残基从SH向NH2发生了分子内转移。在肌质小泡中观察到了这两种类型的反应。产物的比例（可通过其不同的稳定性和吸收光谱来区分）取决于去污剂的有无。对于天然小泡，NH2反应占主导，而对于用十二烷基硫酸钠、八乙二醇单正十二烷基醚或1-O-十四烷基-丙二醇-(1,3)-3-磷酰胆碱溶解的小泡，SH反应占优势。每毫克蛋白质仅0.35毫克十二烷基硫酸钠就足以专门形成二硫代氨基甲酸盐。过量的异硫氰酸荧光素与十二烷基硫酸钠溶解的小泡的几个硫醇基团发生反应。在ATP存在下，异硫氰酸荧光素与天然小泡的结合显著减少。用N-乙基马来酰亚胺完全封闭小泡的SH基团会导致与天然小泡相比，与异硫氰酸荧光素反应慢得多的制剂。测定中八乙二醇单正十二烷基醚或1-O-十四烷基-丙二醇-(1,3)-3-磷酰胆碱会加速N-乙基马来酰亚胺修饰小泡中硫脲的形成，而十二烷基硫酸钠几乎完全阻止其形成。我们的结果支持这样的观点，即高反应性赖氨酰残基附近的一个或几个硫醇基团可能在快速特异性反应中起作用，这种反应仅在完整的天然小泡中观察到。
