CUN. CHEM. 21/11, 1585-1591 (1975) CLINICAL CHEMISTRY, Vol. 21, No. 11, 1975 1585 Effects of Buffers on Aspartate Aminotransf erase Activity and Association of the Enzyme with Pyridoxal Phosphate Robert Rej and Raymond E. Vanderlinde Using purified enzymes of human origin and patients’ sera, we examined factors influencing the in vitro asso- ciation of pyridoxal phosphate with aspartate amino- transferase (EC 2.6.1.1). The rate of association was markedly retarded by phosphate buffer In comparison with tris(hydroxymethyl)aminomethane or six other buff- ers. Pyridoxal phosphate at an incubation concentration of 130 imol/lfter reactivated the entire apoenzyme por- tion of an apoenzyme/holoenzyme mixture within 5 mm in tris(hydroxymethyl)aminomethane; in contrast, less than 20% was associated during 15 mm in phosphate. Activity measured in tris(hydroxymethyl)aminomethane- buffer without exogenous pyridoxal phosphate was 4% greater than that in phosphate and was slightly in- creased by Increasing the pH of the assay mixture from 7.5 to 8.0. Aspartate in the incubation medium did not retard the stimulation in tris(hydroxymethyl)amino- methane buffer.While the magnitude of stimulation var- ied greatlyamong sera, a consistent mean stimulation of 30% for groups of sera with normal activities was found when asparate at 125 mmol/llter, 2-oxoglutarate at 6.7 mmol/liter and tris(hydroxymethyl)aminomethane at 90 mmol/liter were used, an increase over the 16% with phosphate buffer [Clin. Chem. 19, 92 (1973)]. Ab- sorbance spectra suggest pyridoxal phosphate exists as the Schiff base of tris(hydroxymethyl)aminomethane or aspartate, or both, under conditions of assay incubation (without addition of 2-oxoglutarate). Nonenzymatic ca- talysis of the reaction by pyridoxal phosphate alone or a formation of a protein/pyridoxal phosphate adduct was discounted with use of D-asparate substrates. In 1955, Karmen (1) introduced the malate dehy- drogenase/NADH-coupled assay for measurement of AspAT’ activity in serum buffered with a phosphate buffer, pH 7.4. There have been numerous later mod- ifications, kit adaptations, and “optimizations” of Division of Laboratories and Research, New York State Depart- ment of Health, Albany, N. Y. 12201. Portions of this paper were included in a presentation at the 26th National Meeting of the American Association of Clinical Chemists, Las Vegas, August 18-23, 1974 (Paper 172, Clin. Chem. 20, 887 [1974J). 1 Nonstandard abbreviations used: AspAT, aspartate amino- transferase (EC 2.6.1.1), L-aspartate:2-oxoglutarate aminotransfer- ase (formerly known as glutamic-oxalacetate transaminase [GOTJ); malate dehydrogenase (EC 1.1.1.37), L-malate:NAD oxi- doreductase; glutamate dehydrogenase (EC 1.4.1.3), L-glutamate: NAD(P) oxidoreductase (deaminating); Tris, tris(hydroxy- methyl)aminomethane; Tricine, N-tris(hydroxymethyl)-methyl- glycine; HEPES, N-2-hydroxethylpiperazine-N’-2-ethanesulfonic acid; TAPS, tris(hydroxymethyl)methylaminopropane sulfonic acid; pyridoxal-P, pyridoxal-5-phosphate. Received May 30, 1975; accepted July 11, 1975. this technique (2-12), most retaining the use of phos- phate buffer. While there has been some advocacy of the use pf other buffer systems, most notably Tris (6, 8, 10), these have been largely based on arguments for NADH stability (6, 13) rather than upon the ef- fects on AspAT activity itself. Nisselbaum (14) ob- served inhibition of mitochondrial AspAT by phos- phate buffers, and Scardi et al. (15, 16) described the inhibition of apo-AspAT reassociation with pyridoxal phosphate (pyridoxal-P) by this anion. This latter finding is of particular interest, for in our initial stud- ies (17), which showed an average 16% stimulation of serum AspAT activity due to pyridoxal-P, we used a 1-h incubation in the presence of 89 mmol of phos- phate, 134 mmol of aspartate, and 26.8 imo1 of pyri- doxal-P per liter. These studies have been since con- firmed by other laboratories (18-20). The difficulties of such long incubations in routine clinical enzyme laboratories seriously detract from the practicality of such additions of pyridoxal-P. We speculated at the time that in the reaction mixture, phosphate retards the reassociation of pyridoxal-P and apo-AspAT. To investigate the relationships further, we have examined the use of Tris and other buffers in the continuous kinetic malate dehydrogenase/NADH- coupled assay of AspAT activity in serum. Materials and Methods Purified cytoplasmic AspAT was prepared from human erythrocytes or liver by essentially the meth- od of Rej et al. (21). When liver was used as a source, mitochondrial AspAT was removed by ion-exchange chromatography on CM-Sephadex C-50. This materi- al was judged to be a single cytoplasmic isoenzyme by polyacrylamide gel (22) and cellulose acetate electro- phoresis (Rej and Vanderlinde, unpublished). A mix- ture of apo-AspAT and holo-AspAT was prepared by adding L-aspartate (to 83 mmol/liter) and sodium phosphate (to 60 mmolfliter), pH 7.5, to the AspAT solution. This mixture was incubated at room tem- perature for 30 mm and was then thrice dialyzed against 15 volumes of 25 mmol/liter phosphate buff- er, pH 7.5, at 4 #{176}C. This resulted in a material which was approximately 25% apo-AspAT, judged by a 1-h incubation of the final product with 200 tmol of pyri- doxal-P per liter of preparation. This material was diluted with albumin solutions or distilled water for use in the assay. Patients’ sera were selected without