Photochemistry and Photobiology, 1996, zyxwvuts 63(6): 742-745 zyxwvut Symposium-in-Print Chemiluminescence Triggered by Hydrolase Activity in a Horseradish Peroxidase/H,O,-Coupled Assay* Ana Campait, Andrea de Castro Andrade' and Luiz Henrique Catalani2 'Faculdade de Cigncias FarmacButicasand zlnstituto de Quimica, Universidade de zyxwvu SBo Paulo, SBo Paulo, Brasil Received 18 October 1995; accepted 31 January 1996 ABSTRACT zyxwvutsrq 2-Methyl-1-propenyl acetate (MPA) was hydrolyzed by wheat lipase, pancreatic lipase, hydrolases and lipopro- tein lipase from serum in a reaction that produces 2- methyl-1-propenol.The latter was subsequently oxidized by horseradish peroxidase (HRP)M[,02/02 yielding trip- let acetone that emits visible light. The integrated light emission was proportional to the units of lipase or vol- ume of serum present. When pancreatic lipase was as- sayed, light emission was observed in the presence of bile salt as surfactant and chlorophyll zyxwvutsrq a as light sensitizer. The potential application of this reaction in determina- tions of hydrolaseAipase activities is discussed. INTRODUCTION zyxwvutsrq The development of chemiluminescent and bioluminescent assays designed for research and clinical routine purposes are highly desirable given that they are potentially simple, sensitive and that the number of chemiluminometers in lab- oratories is now increasing (1,2). Many kits of chemilumi- nescence immunoassays have already been adopted in clin- ical routines. The development of homogeneous assays that require no separation step has attracted increased attention (3,4). However, chemiluminescent assays for metabolites or enzymes are effectively still to be introduced. Chemiluminescent assays have improved the determina- tion of several substances of clinical interest. The assays described for the measurements of cholesterol (5), glucose (6), lactate and lactate dehydrogenase (7) set the example. Of further interest is the development of sensitive assays for substances whose current available methodology is impaired. This is the case for lipase (8). Despite its value to clinical medicine and biological research, no simple and efficient methodology is available for its routine determination. The main problems associated with the available methodology are long incubation times, unstable and nonreproducible sub- *This paper is dedicated to the memory of Giuseppe Cilento. tTo whom correspondence should be addressed at: Faculdade de CiEncias Farmac&uticas,Universidade de SZo Paulo, SBo Paulo, CEP 05508-900, SP, Brasil. Fax: (0055-11) 8132197. zyxwvuts 0 1996 American Society for Photobiology 0031-8655/96 zyxwvutsrq $S.OO+O.OO strates, requirement of very sensitive devices and lack of discrimination between active and inactive enzyme (9). Here we introduce a chemiluminescent assay by using specific hydrolases in the production of a substrate of horse- radish peroxidase (HRP).$ The HRP-catalyzed oxidation of several a-methine carboxylic acids and aldehydes and their chemiluminescence have been studied by Cilento and co- workers. Among these, the most extensively studied was the HRP-catalyzed oxidation of isobutyraldehyde (IBAL) (10- 15). The HRP cycle initiates when the ferriheme from native HRP either reacts with the peracid, contaminating the sub- strate solution, or with added HzOz (14). This reaction pro- duces compound I that catalyzes the hydrogen abstraction of the enol form of IBAL (13,14). Phosphate buffer is neces- sary for the HRP-catalyzed oxidation of IBAL because it accelerates the rate of IBAL enolization (14,15). Compound I1 formed in the latter step is also catalytically active and regenerates to the enzyme native form. Addition of molec- ular oxygen to the free radical formed by the HRP cycle yields a supposed 1,2-dioxetane intermediate, which decom- poses into triplet acetone and formic acid. Acetone phos- phorescence can be detected with high efficiency. Further- more, the chemiluminescence of this reaction can be en- hanced by electronic energy transfer to acceptors with high quantum yield of fluorescence such as sodium 9,lO-dibro- moanthracene sulfonate (lo), xanthene dyes (1 1) and chlo- rophyll a (1 2). Here we use three types of hydrolases (wheat lipase, pan- creatic lipase and serum hydrolases enriched or not by li- poprotein lipase, LLP) to hydrolyze 2-methyl- 1 -propenyl ac- etate (MPA). The product, which is the enol form of IBAL, is subsequently oxidized by the HRP/H,O,/O, system, pro- ducing chemiluminescence. MATERIALS AND METHODS The MPA was synthesized according to Bedoukian (16). The iden- tity and purity was evaluated by 'H-NMR. Hydrogen peroxide, $Abbreviations: HRP, horseradish peroxidase; IBAL, isobutyralde- hyde; LLP, lipoprotein lipase; MPA, 2-methyl- 1-propenyl acetate; SDS, sodium dodecyl sulfate. 742