ABSTRACT
We have developed a highly sensitive, simple, and practical enzymatic assay by N-(9-acridinyl)maleimide (NAM) fluorometry. An enzymatic method detects the special component among the analogues in living tissue with a simple procedure, and gives different information from that obtained by chemical and physical methods. We found that NAM formed an intensive fluorescent complex with 10−9–10−12 mole of low molecular sulfhydryl compounds released from enzyme reactions, such as reduced glutathione and oxidized glutathione, coenzyme A, or thiols. We have applied the method to the assay of lipid hydroperoxide and carnitine, and the enzyme activities of cholinesterase, carnitine acetyltransferase, glutathione peroxidase and glutathione reductase. NAM fluorometry can be applied to the assay of many compounds by connecting glutathione peroxidase and glutathione reductase, which were key enzymes for enzymatic assay with the other oxidoreductases. The combination with highly selective enzyme reaction and highly sensitive fluorometry was an ideal analytical method for natural products. Moreover using high-performance liquid chromatography together with NAM fluorometry raised the sensitivity more than 100 times.