Stopped-flow peroxidase reactions

The detailed mechanism of P aeruginosa CCP has been studied by a combination of stopped-flow spectroscopy (64, 65, 84, 85) and paramagnetic spectroscopies (51, 74). These data have been combined by Foote and colleagues (62) to yield a quantitative scheme that describes the activation process and reaction cycle. A version of this scheme, which involves four spectroscopically distinct intermediates, is shown in Fig. 10. In this scheme the resting oxidized enzyme (structure in Section III,B) reacts with 1 equiv of an electron donor (Cu(I) azurin) to yield the active mixed-valence (half-reduced) state. The active MV form reacts productively with substrate, hydrogen peroxide, to yield compound I. Compound I reacts sequentially with two further equivalents of Cu(I) azurin to complete the reduction of peroxide (compound II) before returning the enzyme to the MV state. A further state, compound 0, that has not been shown experimentally but would precede compound I formation is proposed in order to facilitate comparison with other peroxidases. 

Enzyme-Immunoassay. Fish tissue samples for testing were cut into uniform 3mm thick slices with parallel razor blades mounted on a handle. Four discs were then punched out from each slice with a stainless steel borer, 3-mm in diameter, and each disc was placed in a well of a 96-well polystyrene microtiter plate (Flow Laboratories, Inc., Hamden, CT). Samples were washed once with 0.2 ml Tris buffer. After the wash solution was aspirated, each sample was fixed in 0.2 ml of 0.3% H O -methanol fixative for 30 min. at room temperature. Samples were then transferred to clean wells and 0.2 ml of a 1 100 dilution of sheep-anti-ciguatoxin-horseradish peroxidase conjugate in Tris buffer was added to each well. The plate was then incubated at room temperature for 1 hr. The sheep-anti-cigua-toxin-horseradish peroxidase was removed by aspiration, and the tissues were immersed for 5 min. in 0.2 ml Tris buffer. Each sample was transferred to clean wells and incubated for 5 min. at room temperature with 0.2 ml of 4-chloro-l-naphthol substrate. The final steps involved removal of the tissue and addition of 0.015 ml of 3 M sodium hydroxide to stop the enzymatic reaction. Absorbance readings at 405 nm of each well were obtained in the Titertek Multi-skan (Flow Laboratories, Inc., Hamden, CT). 

Ricard, J. and Job, D., 1974, Reaction mechanisms of indole-3-acetate degradation by peroxidases. A stopped-flow and low-temperature spectroscopic study, Eur. J. Biochem. 44 359-374. 

The peroxidase-catalyzed reaction of 3,3 -diaminobenzidrne tetrahydrochloride (DAB) with sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, 4-aminoan-tipyrine and H2O2 can be achieved in a stopped-flow microreactor using photothermal temperature control and equipped with an IR diode laser (Figure 7.10) [68, 69]. The time to reach the end of the reaction in the microchip is half of that in a batch process. 

The function and mechanism of action of peroxides have been the subject of a recent review. The peroxidase activity of ferrihaems continues to be investigated. Deuterioferrihaem peroxide compounds (dpc) formed by reaction of H Og, Bu OOH, and ten peroxybenzoic acids have been characterized. The formation of the dpc from the benzoic acid derivatives is instantaneous. The oxidation of iodide by dpc has been studied using stopped-flow methods, the reactions being first-order with respect to each reactant (kg = 1.6x 10 lmol s ) for all hydroperoxide used at pH >7.75. This result supports the concept that all deuterioferrihaem peroxide compounds are the same oxidized form of deuteriohaem, independent of the nature of the oxidant progenitor. 

Figure 1.2 Repetitive spectoa at 1.25 ms interval during the reaction of a mutant of horse-radish peroxidase (Arg38 - Lys) compound 1 (1.25 iM) with /vaminobenzoic acid (200 pM) at pH 7 and 25 C. Experiments were carried out with a HiTech Scientific Ltd SF-61 stopped-flow spectrophotometer with MG-6000 rapid scanning system by Drs A. T. Smith and R. N. F. Thomley (unpublished observations).

So far, examples to illustrate experimental methods for following the time course of the approach to steady states and of their kinetic interpretation have been restricted to enzymes which do not have a natural chromophore attached to the protein although reference has been made to the classic studies of Chance with peroxidase (see p. 142). Qearly the application of these techniques to the study of enzymes with built in chromophores, such as the prosthetic groups riboflavine, pyridoxal phosphate or haem, contributed considerably to the elucidation of reaction mechanisms. However, the progress in the identification of the number and character of intermediates depended more on the improvements of spectral resolution of stopped-flow equipment than on any kinetic principles additional to those enunciated above. This is illustrated, for instance, by the progress made between the first transient kinetic study of the flavoprotein xanthine oxidase by Gutfreund Sturtevant (1959) and the much more detailed spectral analysis of intermediates by Olson et al. (1974) and Porras, Olson Palmer (1981). 

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