CHEMIJA. 2008. Vol. 19. No. 2. P. 9–12 © Lietuvos mokslų akademija, 2008 © Lietuvos mokslų akademijos leidykla, 2008 Application of immobilized citrate lyase in a bioanalytical system for determination of citrates Asta Kaušaitė 1 , Almira Ramanavičienė 1, 2 , Arūnas Ramanavičius 1, 2 * 1 Center of Nanotehnology and Materials Science, Faculty of Chemistry, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania 2 Laboratory of Immunoanalysis and Nanotechnology, Institute of Immunology of Vilnius University, Molėtų pl. 29, LT-08409 Vilnius, Lithuania * Corresponding author. E-mail: arunas@imi.lt A bioanalytical protocol based on the enzymatic-spectrophotometric micro-method for de- termination of citrate with immobilized citrate lyase (CL) is described. In the design of this spectrophotometric bioanalytical system, L-malate dehydrogenase (L-MDH), oxaloacetate de- carboxylase (OACD) and L-lactate dehydrogenase (L-LDH) were applied as a biological recogni- tion system. Immobilized CL catalysed the conversion of citrate to oxaloacetate and acetate. In the presence of the enzymes L-MDH and L-LDH, oxaloacetate and its decarboxylation product pyruvate were reduced to L-malate and L-lactate, respectively. he concentration of citrate is stoichiometric to the concentration of nicotinamide adenine dinucleotide (NADH) formed in enzymatic reaction. NADH was determined by means of its light absorbance at 340 nm. he maximal diference in light absorbance was detected when CL had been immobilized on porous carbon. If CL had been immobilized on wool ibres or carbon rod the absorbance diference ater 360 min was almost 2 times and 3.2 times lower, respectively, as compared with the re- sults observed when CL had been immobilized on porous carbon. he method proposed here has several advantages if compared with the technique based on dissolved CL. Moreover, this method allows achieving a higher rate of CL-catalysed reaction and to reduce the duration of analysis versus the previously suggested methods. We expect that this method can be employed for determination of other analytes by applying the corresponding enzymes in the design of the bioanalytical system. Key words: bioanalytical chemistry, enzymatic detection, biosensors, citrate lyase, citric acid, citrate INTRODUCTION An important ield of analytical chemistry is determination of biologically active substrates. Here, the most promising are bio- analytical systems that are based on immobilized enzymes [1] or some ainity exhibiting materials [2] because such systems allow to determine a variety of biologically active analytes (e. g., glucose, creatine, citric acid, citrates and many others) [3]. Citric acid and citrates are present in numerous natural products. hey are very important intermediates in the metabolism of plants, animals and microorganisms. In the human body, citrates are metabolised and excreted by the kidney [4]. Citrates are prod- ucts of normal oxidative pathways in the body of mammalians and are normally excreted in the urine [5]. Urinary citrates play an important role in the endogenous inhibiting of the formation of calcium-containing kidney stones [6]. Citrates form stable soluble complexes by chelating calcium [7], directly prevent- ing the crystallization and precipitation of calcium-containing compounds [8]. Moreover, citrates interact with Tamm–Horsfall proteins and inhibit Ca-oxalate crystallization [9]. Low urinary citrate concentrations are found in approximately half of adult patients with renal stones [10]. Citric acid is used in the food and beverage industry to improve and protect lavour and aro- ma, and in cosmetics for pH adjustment. It is a component of many pharmaceuticals and washing powders. Determination of citric acid and citrates has become an important item for biological luids (e. g., serum, urine, plas- ma, seminal, sperm plasma) and foodstufs. Several methods have been proposed for the determination of citrates based on ion-exchange chromatography [11], high performance liquid chromatography (HPLC) [12], capillary electrophoresis [13] and isotachophoresis [14]. hese methods are time-consuming procedures; besides, sample preparation is required in order to separate citric acid from other co-existing tricarboxylic acids. Other approaches based on conductometric or spectrophoto- metric methods [15] sufer from selectivity as they are based on non-speciic reactions with carboxylic acids. he above-men- tioned selectivity problems could be potentially solved by using a high speciicity enzyme such as citrate lyase (CL) [16–19]. Enzymatic methods allow a rapid and reliable determination of several constituents. Enzymes are valuable analytical tools and ofer sensitive and speciic methods for quantitative analysis; especially useful and reliable are bioanalytical systems based on immobilized enzymes [20]. Firstly, the procedure utilizing