Sulpho-N-hydroxysuccinimide activated long chain biotin. A new microtitre plate assay for the determination of its stability at different pH values and its reaction rate with protein bound amino groups.

Biotinamidohexanoic acid N-hydroxysulphosuccinimide ester (N-hydroxysulphosuccinimide activated long chain biotin; sulpho-NHS-LC-biotin) has become an invaluable tool for the biotinylation of protein despite the absence of data concerning its stability and reaction velocity. A convenient, rapid and sensitive assay for this compound has been developed based on the sulpho-NHS-LC-biotin mediated biotinylation of bovine serum albumin following adsorption to the wells of a microtitre plate. Bound biotin was visualized by the sequential use of streptavidin and biotinylated horseradish peroxidase. This assay was used for the determination of the stability of sulpho-NHS-LC-biotin in aqueous solution of different pH values. Hydrolysis half lives were below 15 min at pH values above 8.0, but a pH values below 6.5 they exceeded 2 h. It is suggested, therefore, that biotinylations should be performed with sulpho-NHS-LC-biotin taken from a stock solution, prepared at pH values between 3.0 and 5.8. Reaction velocities with primary amino groups were also investigated by means of this ELISA procedure. As expected, biotinylation proceeds faster at pH 8.0 as compared to 7.2, but the increased reaction rate does not compensate for the decreased hydrolysis half life at the higher pH value. Thus, biotinylation with sulpho-NHS-LC-biotin at near neutral pH values appears to be optimal.