Reaction of fluorescein isothiocyanate with thiol and amino groups of sarcoplasmic ATPase.

Abstract

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.