In-situ monitoring of the interaction of lactate dehydrogenase with NAD ona gold electrode by FT-SERS

The FT-SEWS method was used to observe directly and monitor the combination of dehydrogenases with NAD pre-adsorbed onto the surface of a roughened go ld electrode. At negative electrode potentials, the SEWS spectra of NAD exh ibit significant changes upon the addition of lactate dehydrogenase. The en zyme active site may approach the electrode closely enough to produce sever al strong bands attributable to backbone peptide. The experimental results may be explained by binding of the NAD adenine moiety in a hydrohobic regio n of the enzyme while the nicotinamide group is left exposed near the surfa ce of the electrode. This competitive binding state appears to be strongly dependent on the applied electrode potentials. Comparative experiments were carried out with lactate dehydrogenase and glutamate dehydrogenase. The SE WS of NAD upon binding with lactate dehydrogenase show some features signif icantly different from those in the case of glutamate dehydrogenase. This d ifference may result from the different architectures within binding sites of type-A and type-B dehydrogenases. (C) 1999 Elsevier Science S.A. All rig hts reserved.