420 zyxwvutsr D. zyxwvutsrqpo S. Zhao and F. A. Gomez zyxwvutsrqpon Electrophoresis 1998, 19, 420-426 Dong S. Zhao, Frank A. Gomez Double enzyme-catalyzed microreactors using capillary electrophoresis zyxw ~ Department of Chemistry and Biochemistry, California State University, LOs hge1es9 cA, This work evaluates the concept of a double enzyme-catalyzed microreactor using capillary electrophoresis (CE). Migrating in a capillary under electropho- resis conditions, plugs of substrate and two enzymes are injected separately in buffer and allowed to react. Extent of reaction and product ratios were subse- quently determined by CE. This concept is demonstrated using two model systems: the conversion of adenosine triphosphate (ATP) to adenosine diphos- phate (ADP) and adenosine monophosphate (AMP) by hexokinase (HK, EC 2.7.1.1) and apyrase (APY, EC 3.6.1.5), respectively, in the conversion of glu- cose to glucose-6-phosphate and inorganic phosphate, respectively, and the conversion of nicotinamide adenine dinucleotide, reduced form (NADH), to nicotinamide adenine dinucleotide (NAD) and back to NADH by lactate dehy- drogenase (LDH, EC 1.1.1.27) and glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), respectively, in the conversion of pyruvate to lactate and glucose-6-phosphate (glc-6-P) to 6-phosphogluconate, respectively. These procedures illustrate the use of the capillary as a double microreactor and the ease of quantitation of reaction products under conditions of electrophoresis. 1 Introduction Capillary electrophoresis (CE) with its advanced instru- mentation and unique selectivity has become an impor- tant microseparation technique 11, 21. CE differentiates charged species on the basis of mobility under the influence of an applied electric field. The value of the electrophoretic mobility 1.1 of a species is directly related to its net charge and inversely related to its hydrody- namic drag [3]. CE offers a number of advantages as a separation technique: (i) it requires only small quantities of material; (ii) it is applicable to water-soluble, nonvola- tile, high molecular-weight species in aqueous buffer solution; (iii) it is readily automated and has good repro- ducibility; and (iv) selectivity can be manipulated by the alteration of electrolyte properties such as pH, ionic strength, and electrolyte composition, or by the incorpor- ation of electrolyte additives 121. Recently, the microscale capabilities of CE and the high specificity of enzymes has been realized in biochemical analysis by combining CE and enzyme microreactors in a technique known as electrophoretically mediated microanalysis (EMMA) [ 1-20]. In EMMA, differential electrophoretic mobility is utilized to merge distinct zones of analyte and analytical reagent(s) under the influence of an electric field. The reaction is then allowed to proceed within the region of reagent overlap either in the presence or absence of an applied potential, and the resultant product is transported to the detector under the influence of an electric field 1211. There are ~ Correspondence: Professor F. A. Gomez, Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA (Fax: +213-343- 6490; E-mail: fgomez2@calstatela.edu) Abbreviations: APY, apyrase; glc-6-P, glucose-6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; HK, hexokinase; LDH, lactate dehydrogenase Keywords: Capillary electrophoresis / Enzyme-catalyzed microreactors / Quantitation / In-capillary microreactions two major types of EMMA experiments. In the first (continuous format), a sample of the enzyme is injected onto a capillary that is equilibrated with a buffer con- taining the substrate for the enzyme. In the second (plug-plug format), a sample of enzyme and of substrate are injected separately onto a capillary and subjected to electrophoresis; differential mobility in electrophoresis is used to bring them into contact. This type of format has several distinct differences over the continuous format. First, there is no requirement for a chromogenic or fluorogenic reagent. Second, less reagent is needed since only a plug is required as opposed to filling the capillary and buffer reservoirs. Third, because there is less incubation time in comparison with the continuous format (Le., the enzyme plug is in continuous contact with the substrate-filled capillary), the plug-plug approach is inherently less sensitive. We have recently described the ease of quantitating reac- tion products of in-capillary enzyme-catalyzed microreac- tions using CE [2]. Herein, we outline experiments in which double enzyme-catalyzed microreactions occur in an electrophoresis capillary (Fig. 1). We demonstrate this technique using two model systems. In the first system we examine the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) by hexokinase (HK, EC 2.7.1.1) and apyrase (APY, EC 3.6.1.5), respectively, in the con- version of glucose to glucose-6-phosphate and inorganic phosphate, respectively (Eq. 1). HK is a key enzyme in the glycolysis pathway and catalyzes the zy APY zyxwvu I APY zyxwv I ATP ADP zyxwv 7- AMP HK I glucbse glucos’e-&phosphate inbrganic phosphate phosphorylation of glucose to glc-6-P. This phosphoryl- group transfer reaction consumes a molecule of ATP. In its native form HK has a molecular mass of 100 kDa and consists of polypeptide chains of molecular masses slightly higher than 50 kDa. Our current studies exam- 0 WILEY-VCH Verlag GmbH, 69451 Weinheim, 1998 0173-0835/98/0303-0420 $17.50+.50/0