Mechanical Refrigeration

Mode of access: Internet

POTENTIOMETRIC DETERMINATION OF GLUTATHIONE AND GLUTATHIONE TRANSFERASE-ACTIVITY

A new method of determination of Glutathione Transferase activity (GST) and Glutathione (GSH) in solution has been developed. The determination was performed by detection of ions (H+, Cl-, F-) produced during the GST catalyzed reaction using ion-selective electrodes. The Michaelis constants of GST pi from human placenta toward GSH, 1-Chloro-2,4-dinitrobenzene (CDNB) and 1-Fluoro-2,4-dinitrobenzene (FDNB) obtained potentiometrically were similar to those calculated with the classical spectrophotometric method. The use of pH electrode gave the best results for GST activity (1 pH unit slope per 0.1 unit of enzyme in 1 ml of solution; detection limit of 0.001 unit/ml). Different purification steps of GST pi from human placenta followed by this method gave results comparable to the spectrophotometric one. The best calibration curve for GSH (2 x 10(-5) M detection limit) was obtained following the production of F- ions with a fixed amount of enzyme at a concentration of 0.2 U/ml. A calibration obtained performing the measurements in filtrated human serum showed the same behaviour.[...

AN AMPEROMETRIC NADH BIOSENSOR BASED ON NADH OXIDASE FROM THERMUS-AQUATICUS

A biosensor for the determination of the reduced cofactor nicotinammide adenine dinucleotide (NADH) has been developed using the enzyme NADH oxidase from Thermus aquaticus immobilized on an Immobilon AV membrane. A hydrogen peroxide electrode,was used as the detection system. The NADH electrode showed a monotonous response with near linearity in the 5.10(-7) to 2.10(-5) M range, with a detection limit of 2.10(-7) M. Analytical parameters such as pH, response time and lifetime,vere characterized. The probe showed no change in sensitivity between pH 4.5 and 9.5 and retained 70% of its initial activity after 50 days of storage in buffer. A method for the measurement of lactic dehydrogenase (LDH) activity was developed using the biosensor. The response was linear in the 10-1000 U l(-1) range. The addition of NADH and LDH to serum gave recovery values between 94 and 107%. The determination of LDH in two control sera and in three serum samples was performed with a standard spectrophotometric procedure and the biosensor. The results correlated well.[...