RESUMEN
A sensitive and convenient method is described for estimating superoxide dismutase activity using a photochemical augmentation procedure. This method is applicable to both liquid assays and polyacrylamide gel electropherograms. The flux of superoxide is generated by illuminating a reaction mixture containing dianisidine and riboflavin by either a laser source or light from a fluorescent lamp. The oxidation of dianisidine, as sensitized by riboflavin, is enhanced by superoxide dismutase. The increase is linearly dependent on superoxide dismutase concentration. The photochemical reaction is allowed to proceed uninterrupted for a standardized optimum time and intensity of illumination and then terminated by addition of a buffer, 'finibuf', which stabilizes the chromophoric complex formed. This permits the spectrophotometric absorbance measurements of a number of samples collectively and also eliminates the interruption of illumination with the concomitant requirement of a spectrophotometer for constant recording of the absorbance. This method is of utility to both biochemists and clinicians.
Asunto(s)
Espectrofotometría/métodos , Superóxido Dismutasa/análisis , Tampones (Química) , Compuestos Cromogénicos/análisis , Dianisidina/química , Dianisidina/efectos de la radiación , Electroforesis en Gel de Poliacrilamida , Fluorescencia , Rayos Láser , Oxidación-Reducción , Fotoquímica , Riboflavina/química , Riboflavina/efectos de la radiación , Sensibilidad y Especificidad , Superóxidos/metabolismoRESUMEN
On the basis of previous observations, we attempted to characterize the effects of various products of phospholipid hydrolysis on neutrophil (PMN) respiratory burst activity. We studied the effects of phosphorylcholine (PC) and phosphorylethanoline (PE) on superoxide anion production in PMN and in cell free system. We found that PE but not PC inhibited measured superoxide anion, but that this was not due to inhibition of cellular superoxide generation but to scavenging of generated superoxide anion. Further, utilizing a system based upon the photo-oxidation of O-dianisidine sensitized by riboflavin, we were able to determine that the scavenging effect of PE was not superoxide dismutase (SOD)-like but rather a general scavenging or glutathione (GSH)-like effect. These data underscore the importance of identifying the mechanism of inhibition of superoxide generation by putative inhibitors as being due to a direct cellular effect or to a scavenging property.