VOLTAMMETRIC DETECTION OF AVIDIN AND BIOTIN BY BIOTIN-LABELED WITH CYSTEINE

An avidin-biotin assay was developed from a voltammetric procedure usi ng biotin labeled with cysteine. Mercury(II) as a marker was used to d etect avidin and biotin, because the oxidation wave of mercury decreas es when the cysteine part of labeled biotin(LB) complexes with mercury (II). The formation of the mercury(II)-cysteine complex is suppressed when the LB binds to the biotin site of avidin. Accordingly, the conce ntration of avidin can be estimated from the increasing mercury peak c urrent. Detection of biotin is also carried out by a competitive react ion of biotin and the LB to the binding site on avidin, where the addi tion of biotin decreases the peak current of mercury. Limits of detect ion for avidin and biotin were in the 10(-9) mol/L range. The length o f the spacer between the cysteine and biotin was investigated. It was observed that the strength of binding increased with increasing length of spacer. Size considerations rules out steric influences, so it is suggested that the binding constant depends on hydrophobic interaction s in the binding site.