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Anal. Chem. 1990, 62,1022-1026
Application of Adsorptive Voltammetry To Assess the Stability of Modified Alanine Aminotransferases Jose M. Fernkndez Alvarez,' Ciaran O'Fagain? Richard O'Kennedyp Cormac G . kilt^,^ and Malcolm R. Smyth*J School of Chemical Sciences, Dublin City University, Dublin 9, Ireland, School of Biological Sciences, Dublin City University, Dublin 9, Ireland, and Baxter-Dude AG, Dudingen, Switzerland
Alanine aminotransferase has been stabilized by uslng chemical modification with both bls(lmldates) (of varying length) and succinic anhydride. The voltammetric behavior of the native enzyme and its various modified forms has been studied by using both cycllc dammetry and differentlai puke adsorptlve voltammetry. A distlnctlve accumulation pattern was found for each of the stabilized enzymes at the static mercury drop electrode with respect to the native alanine aminotransferase. Adsorptive voltammetry was demonstrated to be a useful technlque to assess the extent of chemical modWlcatlonof this enzyme, which Is indirectly related to their stabllity for use in blotechnologlcal processes. The use of differential pulse adsorptlve voltammetry, after a preconcentratlon of the enzyme for 300 s at the electrode surface, has yielded a detection limit of 1.0 X lo-@M.
INTRO DUCT10N Enzymes are used in a variety of products and processes encountered throughout biotechnology. I t follows therefore that the catalytic activity of an enzyme is of critical importance. I t is often necessary, however, to enhance the stability of a given enzyme, and a variety of methods have been evolved for this purpose, including the addition of known stabilizing compounds such as glycerol or ammonium sulfate ( I , 2 ) , immobilization of the enzyme onto solid supports or within a gel matrix (3),cross-linking of the protein backbone (4-7), or chemical modification of surface functional groups (8,9). This has been recently reviewed by O'Fagain e t al. (IO)and Mozhaev et al. (11). Not all of these methods are, however, applicable to a particular enzyme. For example, an enzyme used in a clinical diagnostic control must be in free solution, and the use of certain additives may result in unacceptable viscosity or inappropriate salt balance. A variety of methods have been used t o assess the degree of stabilization of catalytic activity conferred by particular modifications. These include comparison of resistance to denaturing salts (such as urea) between native and modified enzymes ( I Z ) , comparison of resistance to thermal denaturation at elevated temperatures ( 1 3 , 1 4 ) ,accelerated storage testing (15),and real-time stability testing (which could take over a year to complete). This paper reports on the application of adsorptive voltammetry for a study of a clinical diagnostic marker, the soluble enzyme alanine aminotransferase [L-alanine: 2-oxoglutarate aminotransferase, EC 2.1.6.2,ALT] (16, 17), which has been stabilized both by cross-linking with bis(imidates) (resulting in charge retention) and by functional group modification with succinic anhydride (resulting in charge reversal). We have recently reported on the use of this technique for the study of a variety of immunological and other protein-substrate interactions (18-21). School of Chemical Sciences. *School of Biological Sciences. Baxter-Dade AG.
EXPERIMENTAL SECTION Apparatus. All enzyme assays were carried out with a PyeUnicam Model SP 8400 double-beam UV/visible spectrophotometer with cells thermostated at 30 "C using an external circulating water bath. Voltammetric experiments were carried out by coupling an EG&G Princeton Applied Research (PAR) Model 303A static mercury drop electrode (SMDE) to a PAR Model 264A polarographic analyzer. Voltammograms were recorded on a Houston Instruments Model 2000 X-Y recorder. Stirring of the solutions was achieved with a PAR Model 305 stirrer. A Ag/AgCl (KCI saturated) reference electrode and a platinum wire auxiliary electrode were employed in the three-electrode cell design. A 0.1 M phosphate buffer (pH 7.1) was used as background electrolyte and deionized water was obtained by passing distilled water through a Milli-Q (Millipore) water purification system. Materials. Beef heart ALT was a gift from Dr. A Posner, Baxter-Dade, Miami, FL. All chemicals used for ALT modification and for voltammetric experiments were supplied by Sigma. The Enzfitter software designed by R. J. Leatherbarrow was obtained from Biosoft/Elsevier, Cambridge CB2 lLA, UK. Procedures. ALT activity was measured by using the International Federation of Clinical Chemistry (IFCC) continuous spectrophotometric method (22). Pyridoxal phosphate was included as an activator (22) in the Tris-alanine buffer, pH 7.5. Purification of the enzyme was based on the method described by Jenkins and Saier for pig heart ALT (17). Modification by bis(imidates) was based on the protocols of de Renobales and Welch (23) and Minotani et al. (24). ALT was treated with bis(imidates) of varying molecular length (dimethyl adipimidate, 0.77 nm; dimethyl pimelimidate,0.92 nm; dimethyl suberimidate, 1.10 nm) Succinic anhydride treatment was an adaption of the procedures of Torchilin et al. (25)and Hollecker and Creighton (26). Accelerated storage tests were carried out at 45,37,33, and 27 "C according to the recommendations of Kirkwood (27) and were analyzed by using the Enzfitter package. For voltammetric studies, the test solution (20 mL) was transferred to the cell and deaerated by passing oxygen-free nitrogen for 15 min (and at least for 30 s before each new scan). Adsorptive voltammetric experiments were carried out by using a medium size drop (area = 0.016 cm2). The accumulation (preconcentration step) was carried out at +0.05 V, and a lapse of 15 s was allowed before scanning the potential in the differential pulse (DP) mode, using a pulse amplitude of 50 mV, a scan rate of 10 mV s-l, and a drop time of 0.5 s. For calibration purposes a preconcentration time of 300 s under stirring (400rpm) was used. Cyclic voltammetric (CV) measurements were carried out by using a scan rate of 100 mV s-l. RESULTS Biochemical Experiments. Measurements of ALT activity following treatment with bis(imidates) ranged from 88% to 92%, while that from the succinic anhydride experiment was approximately 96%, compared to native ALT. All of the enzyme activities recorded during the accelerated storage studies fitted to a first-order exponential decay curve, as shown in Figure 1 for the bis(imidate)-modified ALT enzymes a t 45 "C. This shows that the loss of catalytic activity takes place by a unimolecular mechanism and that neither of the chemical modifications carried out changed the kinetic control of the process.
C 1990 American Chemical Society 0003-2700/90/0362-1022$02.50/0
ANALYTICAL CHEMISTRY, VOL. 62, NO. 10, MAY 15, 1990
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