Pyruvate Carboxylase as a Model for Oligosubstituted Enzyme-Ligand Conjugates in Homogeneous Enzyme Immunoassays

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Abstract

Theoretical models suggest that the detection capabilities of homogeneous enzyme immunoassays can be improved by the use of oligosubstHuted enzyme-ligand conjugates rather than the traditionally used muttlsubstltuted ones. The natural form of pyruvate carboxylase contains four covalently bound biotins (one per subunit) and it can be considered as an oligosubstituted enzyme-biotin conjugate. The enzyme is nearly completely inhibited in the presence of the natural binder for biotin, avidin. When the enzyme is incubated with avidin and free biotin, a competition occurs between the free biotin and the prosthetic group of the enzyme for the avidin. Steep dose-response curves are obtained by relating the observed inhibition to the free biotin concentration. By variation of the amount of avidin or enzyme in the assay, the detection limits of the system can be altered allowing for sensitive determinations over a wide range of biotin concentrations. Such data from real sample analysis of several vitamin supplements are reported.

Original languageEnglish (US)
Pages (from-to)2160-2164
Number of pages5
JournalAnalytical Chemistry
Volume61
Issue number19
DOIs
StatePublished - Oct 1989
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry

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