Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography.
A procedure employing C18 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-Ser108]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 degrees 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 and alkaline pH and for evaluating biotin reagent homogeneity.
Kurosky
A
A
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0645.
Miller
B T
BT
Knock
S L
SL
D000595:Amino Acid Sequence; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013388:Succinimides / Q000737:chemistry*
D010455:Peptides; D013388:Succinimides; C033788:biotinyl N-hydroxysuccinimide ester; D001710:Biotin