The peroxidase-catalyzed aerobic oxidation of a linear aldehyde, leading to the next lower homologue in the triplet state, is accompanied by Norrish type H product ethylene when the substrate is pentanal. This system appears to provide an example of "photobiochemistry without light" because ethylene is a plant hormone and is formed in lipid peroxidation when the recurrent oxidation of linear aldehydes occurs. In this work, we report a study ofthis reaction as a function of aldehyde chain length and show that, when pentanal is the substrate, the reaction is accompanied by the formation of the predicted Norrish type II (5) product ethylene. MATERIALS AND METHODSThe aldehydes (Fluka), HRP (types VI, I, and II; Sigma), and eosine (Merck) were commercially available. Aldehydes were stored under N2 at -20°C. The trimethylsilylenol ethers of pentanal and of butanal were prepared according to Cazeau et al. (6).Unless otherwise stated, HRP type VI was used throughout the work; it was sufficiently pure, as attested to by the reproducibility of results in this and in all previous work, even when subjected to further purification. The absence of detectable amounts of ethylene in the absence of aldehydes (see Results and Discussion) indicates that the HRP used contains no ethylene-producing endogenous substrate.Comparative oxygen consumption by the C2-C6 aldehydes in the presence of HRP necessary for the experiments with eosine was ascertained in a Warburg apparatus. The (slow) rate of oxygen uptake was as follows: C2 < C3 < C4 = C5 = The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.C6. When the trimethylsilylenol ethers were used as substrates, oxygen uptake was measured in a Yellow Springs Instruments model 53 oxygen monitor.The experiments with eosine were done at 40°C in 0.5 M phosphate/0.04 M sodium pyrophosphate buffer, pH 7.4/0.5 M ethanol/10 ,uM eosine/1 x 10-4 M hydrogen peroxide/2 mM, 4 mM, or 8 mM aldehyde; total volume was 3 ml. The reaction was initiated by HRP addition (final concentration, 2 ,uM); under these conditions eosine is stable. Light emission was measured in a Hamamatsu model TUC-767 photon counter provided with a Hamamatsu R-562 photomultiplier tube.The experiments with the trimethylsilylenol ethers of pentanal and of butanal were done at 37°C in 0.01 M Tris buffer, pH 7.4/2 mM substrate/4 ,uM HRP. The reaction was initiated by adding 1 x 10-4 M hydrogen peroxide. The system was incubated for 10 min in a 124-ml round-bottom flask sealed with a Teflon-lined rubber septum.The experiments with butanal and propanal were done at 37°C in 0.5 M phosphate buffer, pH 7.4/0.04 M sodium pyrophosphate/1 x 10-4 M hydrogen peroxide/8 mM aldehyde (freshly distilled). The reaction was initiated by adding 1 X 10-4 M hydrogen peroxide. The incubation (as above) was for 50 min. Regarding hydrogen peroxide use in all these systems, note that hydrogen per...

In the presence of the surfactant hexadecyltrimethyl ammonium bromide (CTAB) a cascade of electronically excited states accompanies the successive steps in the peroxidative metabolization of the strong estrogenic and tumourogenic diethylstilbestrol. Reversing the order by necessity, we report in this first paper results with the metabolites. Exposure of 4-hydroxypropiophenone, Z,Z-dienestrol or E,E-dienestrol to horseradish peroxidase and H2O2 promotes oxygen uptake and spectral alterations. Light emission is observed provided that the surfactant CTAB is present. With the three substrates, 4-hydroxybenzoic acid and a new metabolite, p-benzoquinone, have been identified. With both dienestrol isomers, 1-(4'-hydroxyphenyl)-propan-1-on-2-ol has been identified. In all cases the emission spectrum indicates the presence of several emitters. Possible chemiexcitation routes are pointed out. From the dramatic increase of the emission by enhancers, values as high as 1 x 10(-5) are inferred for the product of the quantum yields of chemiexcitation and energy transfer.

RESUMO

When the synthetic estrogen and tumourogenic compound diethylstilbestrol is exposed to horseradish peroxidase (HRP) and H2O2 in the presence of the cationic surfactant hexadecyltrimethylammonium bromide (CTAB), a burst of oxygen consumption and concomitant light emission are observed. The quinone form of the product is not seen in the absorption spectrum because CTAB strongly catalyses its conversion to Z,Z-dienestrol. The emission spectrum shows several peaks. Total emission is dramatically enhanced by chlorophyll and by xanthene dyes. A key intermediate in chemiexcitation is 4-hydroxypropiophenone. The ability to promote chemiexcitation is retained through various generations of metabolites, giving origin to a cascade of excited states. Since the biological effects of diethylstilbestrol appear to be connected with its peroxidative metabolism, chemiexcitation may eventually prove to be of importance in, for example, toxicity of the drug.

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