Characteristics of chemiluminescence observed in the horseradish peroxidase-hydrogen peroxide-tyrosine system

Electrolysis or horseradish peroxidase (HRP)-catalyzed oxidation of tyrosin e and bityrosine in aqueous solution at pH 7.4 resulted in light emission i n the visible region. Electrolysis of tyrosine emitted light which peaked a t 490 nm and was almost completely quenched by superoxide dismutase (SOD), while emission by bityrosine peaked at 530 nm. In the HRP-H2O2-tyrosine sys tem the oxidation-reduction of tyrosine emitted light with two prominent pe aks, 490 and 530 nm, and was not quenched by SOD, The phenoxyl neutral radi cal of the tyrosine in HRP-H2O2-tyrosine system was detected by electron sp in resonance (ESR) spectrometry using tert-nitrosobutane as a spin trap; th e spin adduct was found to adhere to the HRP molecule during the enzymatic reaction. Further, bityrosine was detected in the HRP-H2O2-tyrosine reactio n system. Changes in absorption spectra of HRP and chemiluminescence intens ities during HRP-catalyzed oxidation of tyrosine suggest that for photon em ission compound III is a candidate superoxide donor to the phenoxyl cation radical of tyrosine on the enzyme molecule. The luminescence observed in th is study might be originated from at least two exciplexes involved with the tyrosine cation radical (Tyr(.+)) and the bityrosine cation radical (BT.+) as compound III + Tyr(.+) --> [Tyr(.+)O(2)(.-)]* + HRP, compound III + BT.+ --> [BT.+O2.-]* + HRP, and [Tyr(.+)O(2)(.-)]* --> hv (490 nm), [BT(.+)O2 .-]* --> hv (530 nm), where [Tyr(.+) O-2(.-)]* and [BT.+ O-2(.-)]* are exciplexes. (C) 1999 Acade mic Press.