Anal. Chem. 1982, 5 4 , 1991-1997
1991
Tyrosine-Selective Enzyme Probe and Its Application Jeno Havas’ and George G. Gullbault* Department of Chemistry, Universiv of
New Orleans, New Orleans, Louisana 70 148
A potentlometrlc L-tyrosine-selectlve probe Is described for the dlrect determlnatbn of tyrosine concentration in blologlcal flulds and foods. The sensor element of the probe, based on a carbon dloxlde senrltlve gas sensor, Is a layer containing ImmoMllzed L-tyrosine decarboxylase enzyme and apoenzyme covalently bound on the surface of a piece of small lntestlne from a pig. The electrochemlcai characterlstlcs of the probe were studied. The lmnnoblllzed enzyme layer Is stable for 3-5 months and exhibits ai h e a r response to L-tyroslne over the 2.6 X to 4 X mol/L range. No Interference from L-amino acids and D-tyrosine was observed. The optimum condltlons for the measurement and for storage of the Immoblllzed enzyme layer were evaluated.
The concentration of some amino acids (e.g., phenylalanine, tyrosine) in biological Jluids and tissues is a useful indicator of various disturbances in the organism (2-4), and the quantity of certain amino acids) (e.g., lysine) in foods is characteristic of their quality (5,6).hi both fields, a rapid method is needed that enables determinations to be performed directly, selectively, and, if possible, continuously. Many methods have been described for the analysis of amino acids (7,8),but only one, the method using the amino acid selective probe, is suitable for this task. The amino acid selective probe is an electroanalytical device comprising an immobilized enzyme layer and a gas sensor or anion-selective electrode. Detailed information on the production and application of molecule-selective probes can be found in the works of Guilbault (9) and Havas (10). Many of the numerous organic molecule selective probes developed during the last decade (9) are of the amino acid selective kind (8, 11-203). The first tyrosine probe was described by Guilbault and Shu (15). In 1974 Berjonneau et al. (18) and in 1975 Calvot et al. (20) also described a tyrosine probe based on a carbon dioxide sensor covered with an immobilized L-tyrosine decarboxylase enzyme layer. Unfortunately, these probes have a short lifetime (10 h to a few days), limited measuring range to 1.5 X mol/L), and long response time (5-8 min). However, the greatest liability is that the enzyme layer if3 mechanically too unstable and cannot be reproduced consistently. Production of the tyrosine-selective probe for longer use and suitable sensitivity not only has technological difficulties but also the drawback of the comparative activity of tyrosine decarboxylase enzyme being too low. For this reason we attempted to set up the tyrosine-selective probe by applying the comparatively simpler structure of decarboxylase apoenzyme. Preliminary experiments, using some of the known technology (cross-linking of the enzyme and BSA directly on the gas-permeable membrane, immobilization in collagen membrane), produced unfavorable results: The immobilized enzyme layer easily separated from the Teflon membrane of the pcO;sensor, or rather, the activity of the immobilized layer was immeasurably minute. Therefore, for the production of Present address: RADELKIS Electrochemical Instruments Co., Research and Development Department, H-1300 Budapest, P.O.B. 106, Hungary. 0003-2700/82/0354-1991$01.25/0
the tyrosine-selective probe we chose the technology successfully applied earlier (24)for achieving a glucose-selective probe, Le., the cross-linking of the enzyme and the BSA on the boundary layer of the small intestine membrane containing amino groups. This paper describes an L-tyrosine-selective probe for the determination of tyrosine concentration in biological fluids and foods. The sensor element of the probe, based on a carbon dioxide sensitive, gas sensor is a layer containing immobilized L-tyrosine decarboxylase enzyme or apoenzyme covalently bound onto the surface of a piece of small intestine from a pig. Optimization factors for immobilization of decarboxylase enzymes were investigated. Electrochemical characteristics of the probe, as well as the optimum conditions for L-tyrosine determination, were also studied. The function of the tyrosine probe is based on the decarboxylation of L-tyrosine by the enzyme. During the reaction, liberated carbon dioxide is measured continuously by the carbon dioxide sensor.
EXPERIMENTAL SECTION Apparatus. The carbon dioxide sensors used were a product of Universal Sensors (type: US 3) and of Radiometer, Denmark (type: E-5036),fitted with a Teflon membrane (Universal Sensors, thickness 15 pm; Radiometer, type DG02). The sensor response was measured with a Radiometer digital acid-base analyzer pH M72 Mk-2 and recorded with a Radiometer Servograph REC 61 recorder. The calibration of the sensor was performed with hydrogen carbonate solution added into a buffer (if we add hydrogen carbonate solution to a buffer solution with a pH
