On the application of the Clark oxygen electrode to the study of enzyme kinetics in apolar solvents: The catalase reaction
Por:
Escobar L., Salvador C., Contreras M., Edgardo Escamilla J.
Publicada:
1 ene 1990
Resumen:
A method for recording O2 concentrations in nonconducting organic media with the Clark oxygen electrode was developed. Spontaneous oxidation of Na2S2O4 and the enzymatic reduction of NaBO3 or H2O2 by bovine liver catalase trapped in hydrated micelles of dioctyl-sulfosuccinate (AOT)/toluene were used as model systems. O2 titration with the above systems showed that air-saturated 1.6 m H2O/0.2 m AOT/toluene media contain seven times more O2 (1.4 mm) than aqueous solutions (0.2 mm). The measured Km values of catalase for NaBO3 and H2O2 in organic media were Kmov = 15 and 17 mm, respectively, whereas in aqueous buffer the values were 45 and 54 mm. In the toluene media, catalase activity increased with the W0 (H2O/AOT molar ratio) of the micellar preparation, reaching maximal activity at W0 = 10-12; under this condition, the catalytic center activity (Kp) of H2O2 was 7 × 106 min-1, similar to that obtained in the aqueous buffer (H2O2 = 7 × 106 min-1). It was found that the optimal pH for catalase in toluene media (pH 8.0) was shifted 1.0 unit compared to that in the aqueous buffer (pH 7.0). On the other hand, catalase was severely inhibited by NaN3 in both media. Thus, polarography based on the Clark oxygen electrode seems to be an easy, rapid, and sensitive technique for studying enzyme reactions consuming or evolving O2 in apolar media. © 1990.
Filiaciones:
Escobar L.:
Unidad de Investigación Biomédica, Centro Médico Nacional, Instituto Mexicano del Seguro Social, A.P. 73032, 06760 México, D.F., Mexico
Salvador C.:
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, A.P. 70-242, 04510 México, D.F., Mexico
Contreras M.:
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, A.P. 70-242, 04510 México, D.F., Mexico
Edgardo Escamilla J.:
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, A.P. 70-242, 04510 México, D.F., Mexico
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