Simple ultraviolet spectrophotometric method for the determination of serum guanase activity

Abstract

We describe a simple, kinetic method for the determination of serum guanase activity that involves enzymatic coupling to xanthine oxidase and measurement of the rate of uric acid formation by spectrophotometric monitoring of the absorption at 300 nm. At this wavelength, the absorption of uric acid is about 80% of its maximal absorption at 293 nm, but the difference in molar extinction coefficient between guanine and uric acid is similar (9,000 at 293 nm vs 8,400 at 300 nm). There are three advantages to the use of the higher wavelength: first, the absorption of serum proteins is only one third of the absorption at 293 nm resulting in a significant reduction in noise level. Second, the lower absorption of serum proteins allows increasing sensitivity of the assay by increasing the amount of serum in the reaction mixture. Third, the higher wavelength allows the use of automated centrifugal analyzers that are generally not designed for measurements below 300 nm. The between-day coefficient of variation was 5.8% (n = 27) at an activity of 17 U/L and 8.2% at an activity of 2 U/L. The reference range for 50 sera from males and females was 0.4 to 1.8 U/L (n = 50; mean +/- 2SD). The method is linear to 40 U/L.