Abstract
A procedure employing C 18 reversed-phase high-performance liquid chromatography (HPLC) is described for evaluating the kinetics of biotinylation of specific residues of peptides after reaction with N-hydroxysuccinimide esters of biotin. Utilizing this HPLC method we determined the observed pseudo-first-order reaction rate constant ( k′ 1) of biotinylation of lysyl residues in two model peptides [biotinyl-Ser 108]ProA-egg laying hormone (108-121) and pGlu-Lys-Trp-Ala-Pro, to be 1.22 · 10 −2 s −1 and 1.08 · 10 −2 s −1, respectively in 0.05 M sodium phosphate buffer, pH 8.2, at 25°C. The respective reaction half-lives of the two peptides were 57 s and 64 s. In addition, HPLC analytical methods were established for determining the time-course of hydrolysis of biotinylating reagents at acidic alkaline pH and for evaluating biotin reagent homogeneity.
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