<Originals> An analysis of the reaction between immobilized LD and NADH

We tried to analyse the reaction between LD and NADH using a FIA (flow injection analysis) system involving an immobilized enzyme column and chemiluminescence. Peak resolution was determined for NADH utilizing two methods: a sequential LD/NADH oxidase (OD) column reactor, LD column on the upstream and NADH oxidase column on the downstream, and a sequential NADH OD/LD column, NADH OD column on the upstream and LD column on the downstream. The former method resulted in broad peaks, whereas the latter method produced narrower peaks. When lactate was determined, the peak widths obtained using the sequential LD/NADH OD column reactor became remarkably broad. In contrast, a coimmobilized LD/NADH OD column produced narrow peaks and an eight-fold increase in chemiluminescent intensity over the sequential LD/NADH OD column. This phenomenon was not limited to LD and was reproducible with the following dehydrogenase : malate dehydrogenase, glutamate dehydrogenase, and glucose-6-phosphate dehydrogenase. Those results show that because LD and NADH OD are located in close proximity in the coimmobilized LD/NADH OD column, NADH produced by the LD reaction is accessed by NADH OD as soon as it is found on the surface of LD. In consequence, the whole process proceeds without retardation, and the peaks on the chart become satisfactorily sharp. Therefore, when a dehydrogenase is utilized as an analytical reagent, a coimmobilized dehydrogenase/NADH OD column works effectively