Determination of Aldehyde Dehydrogenase Activity by Fluorescence Spectrophotometer

Abstract

A method for the determination of aldehyde dehydrogenase activity by fluorescence spectrophotometer was established. The precipitate of yeast was added by three volumes of phosphate buffers after centrifugation. Ultrasonic fragmentation of cells is under ice-cooling condition. The supernatant was collected by centrifugation as a crude enzyme solution of aldehyde dehydrogenase after sonication. The effects of pH and temperature on enzyme activity were investigated. Ammonium sulfate precipitation method and polyethylene glycol (PEG) precipitation method were alternative to purify the crude enzyme, and the effect of PEG precipitation was better. The purification factor of the enzyme protein was 2.32. NADH (nicotinamide adenine dinucleotide) solutions were scanned to determine the wavelength of fluorescence detection: excitation wavelength at 345 nm and emission wavelength of 460 nm. The fluorescence spectrophotometer detection limit was 0.2 mg/L, the linear range was 0.75-60 mg/L and the correlation coefficient was 0.995. The recoveries were 95.62% -99.21% with the relative standard deviations (RSD) of 0.60%-0.98% (n=6). This method was utilized to detect the activity of aldehyde dehydrogenase and the results were compared with the results of UV spectrophotometry. The results indicate that fluorescence spectrophotometer is a suitable method for the detection of aldehyde dehydrogenase enzyme activity.