Luminol chemiluminescence induced by immobilised xanthine oxidase Original Research Article

The properties of xanthine oxidase, immobilised on controlled-pore glass, were investigated and applied in assays for hypoxanthine and superoxide dismutase. Phosphate buffer (pH 7.8) was passed through a glass reactor packed with immobilised enzyme, immediately mixed with 1.0 × 10−4 M luminol in carbonate buffer at pH 10.0 and CL measured. CL was enhanced 14-fold by EDTA and 57-fold by Fe(II)–EDTA. For soluble enzyme, injected into a similar FIA manifold, CL was enhanced to a lesser extent either by iron(II) or EDTA or iron(II)–EDTA. CL is enhanced more using dialysed enzyme than with crude enzyme. CL signals are initially high due to rapid reaction between iron(II) and oxygen, but the steady emission used for measurement results from the superoxide-driven Fenton reaction, generating hydroxyl radicals that oxidise luminol which then reacts with enzymatically produced superoxide. The calibration for hypoxanthine was linear (r2 = 0.9834, n = 7) up to 17 μM: CL/mV = 0.0371[HX]/μM − 0.0142. The R.S.D. (n = 4) was 1.69% at 8.24 μM and the apparent Km ≥ 16 μM, ≥12× greater for immobilised enzyme than for the soluble xanthine oxidase. The chemiluminescence decreased with increased superoxide dismutase (SOD) concentration (IC50 = 1.2 μg ml−1 and detection limit = 0.25 μg ml−1) and it was shown to decrease to a lesser extent for catalase.