Analytical Sciences

Voltammetric Homogeneous Binding Assay of Biotin without a Separation Step Using Iminobiotin Labeled with an Electroactive Compound

Kazuharu SUGAWARA,* Naoto KAMIYA,* George HIRABAYASHI,* and Hideki KURAMITZ**

*Faculty of Education, Gunma University, Maebashi, Gunma 371-8510, Japan
**Department of Environmental Biology and Chemistry, Faculty of Science, Toyama University, Toyama 930-8555, Japan

Avidin, which is one type of glycoprotein, has a strong affinity with biotin (K_a = 10¹⁵ M^-1). Iminobiotin also forms a complex with avidin (K_a = 10⁸ M^-1 at pH 9.5). The avidin-iminobiotin complex changes to the avidin-biotin complex in the presence of biotin because of the difference of the binding constant to avidin. In this study, the interaction between avidin and iminobiotin labeled with an electroactive compound was investigated by voltammetry. After avidin and the labeled iminobiotin (LI) were incubated in 0.1 M phosphate buffer (pH 7.0), the peak currents of LI were measured in various concentrations of biotin. The peak currents increased with increasing the concentration of biotin. Thus, this observation indicates the formation of avidin-biotin complex. On the other hand, the formation of avidin-iminobiotin complex depended on the pH of the solution. LI combines with the avidin at pH 5.6 - 8.9 and dissociates at pH 4.6.

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