Kinetic and titration methods for determination of active site contents of enzyme and catalytic antibody preparations.
Kinetic characterization of enzymes and analogous catalysts such as catalytic antibodies requires knowledge of the molarity of functional sites. Various stoichiometric titration methods are available for the determination of active-site concentrations of some enzymes and these are exemplified in the second part of this article. Most of these are not general in that they require the existence of certain types of either intermediate or active-site residues that are susceptible to specific covalent modification. Thus they are not readily applicable to many enzymes and they are rarely available currently for titration of catalytic antibody active sites. In the first part of the article we discuss a general kinetic method for the investigation of active-site availability in preparations of macromolecular catalysts. The method involves steady-state kinetics to provide Vmax and Km and single-turnover first-order kinetics using excess of catalyst over substrate to provide the analogous parameters k(obs)lim and K(m)app. The active-site contents of preparations that contain only active catalyst (Ea) and inert material (Ei) may be calculated as [Ea](T) = Vmax)/k(obs)lim. This is true even if nonproductive binding to E(a) occurs. For polyclonal catalytic antibody preparations, which may contain binding but noncatalytic material (Eb) in addition to Ea and Ei, the significance of Vmax/k(obs)lim is more complex but provides an upper limit to E(a). This can be refined by consideration of the relative values of Km and the equilibrium dissociation constant of EbS. Analysis of the Ea, Eb, Ei system requires the separate determination of Ei. For catalytic antibodies this may be achieved by analytical affinity chromatography using an immobilized hapten or hapten analog and an ELISA procedure to ensure the clean separation of Ei from the Ea + Eb mixture.
Brocklehurst
K
K
Laboratory of Structural and Mechanistic Enzymology, School of Biological Sciences, University of London, United Kingdom. kb1@qmw.ac.uk
Resmini
M
M
Topham
C M
CM
D000906:Antibodies / Q000737:chemistry; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D002384:Catalysis; D004798:Enzymes / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D013997:Time Factors
D000906:Antibodies; D004798:Enzymes