Involvement of lactoperoxidase in the peroxidative degradation of serotonin: a potential pathway for indolaminergic melanin formation

The peroxidase-catalyzed degradation of 5-hydroxytryptamine (serotonin) was studied using rapid scan or conventional spectrophotometry for detection of one-electron conversions of enzyme compounds I, II and III. The spectral changes of serotonin during oxidation and spectral and bleaching properties of reaction products were examined. The results of the investigation clearly indicate the ability of serotonin to function as an electron donor substrate for animal peroxidases.     

Studies of the induction of dominant lethals and translocations in male mice after chronic exposure to microwave radiation

Male C3H mice were exposed to 100 W m-2 of 2.45 GHz continuous-wave microwave radiation for 6 h per day for a total of 120 h over an 8-week period. The exposure level was chosen so that the specific energy absorption rate (SAR) would be approximately equal to the level of 4 W kg-1 which is considered by a number of organizations to be a threshold for adverse biological effects. At the end of the treatment period the mice were mated with a different group of (C3H x 101) F1 hybrid females each week for the following 8 weeks. There was no significant reduction in pregnancy rate, preimplantation survival or postimplantation survival in the exposed group compared to sham-exposed controls. At the end of the mating period a cytogenetic analysis was carried out of meiotic chromosome preparations of testicular tissue, thus sampling cells that were stem cell spermatogonia during the treatment regime. The results showed no difference in the frequency of reciprocal translocations between the sham and treated groups, or in the frequency of cells with autosome or sex chromosome univalents. Low levels of fragments and exchanges were found in both groups. It is concluded that there is no evidence in this experiment to show that chronic exposure of male mice to 2.45 GHz microwave radiation induces a mutagenic response in male germ cells. This conclusion is in agreement with the observations of Berman et al. (1980), who reported a lack of male germ cell mutagenesis after repetitive or chronic exposure of rats to 2.45 GHz.     

Spectroscopic and kinetic properties of the oxidized intermediates of lignin peroxidase from Phanerochaete chrysosporium

Stopped-flow rapid scan techniques were used to obtain a spectrum of nearly homogeneous lignin peroxidase compound I (LiPI) under pseudo-first order conditions at the unusually low pH optimum (3.0) for the enzyme. The LiPI spectrum had a Soret band at 407 nm with approximately 60% reduced intensity and a visible maximum at 650 nm. Under steady-state conditions a Soret spectrum for lignin peroxidase compound II (LiPII) was also obtained. The Soret maximum of LiPII at 420 nm was only approximately 15% reduced in intensity compared to native LiP. Transient state kinetic results confirmed the pH independence of LiPI formation over the pH range 3.06-7.39. The rate constant was (6.5 +/- 0.2) x 10(5) M-1 S-1. Addition of excess veratryl alcohol to LiPI resulted in its reduction to LiPII with subsequent reduction of LiPII to the native enzyme. Reactions of LiPI and LiPII with veratryl alcohol exhibited marked pH dependencies. For the LiPI reaction the rate constants ranged from 2.5 x 10(6) M-1 S-1 at pH 3.06 to 4.1 x 10(3) M-1 S-1 at pH 7.39; for the LiPII reaction, 1.6 x 10(5) M-1 S-1 (pH 3.06) to 2.3 x 10(3) M-1 S-1 (pH 5.16). These single turnover experiments demonstrate directly that the pH dependence of these reactions dictates the overall pH dependence of this novel enzyme. These results are consistent with the one-electron oxidation of veratryl alcohol to an aryl cation radical by LiPI and by LiPII.     

Peroxidase-catalyzed formation of triplet acetone and chemiluminescence from isobutyraldehyde and molecular oxygen

It has been established that the horseradish peroxidase/O2/isobutyraldehyde (IBAL) system leads to triplet acetone and formic acid formation followed by phosphorescence of the triplet acetone (see, for example, Bechara, E.J.H., Faria Oliveira, O.M.M., Durán, N., Casadei de Baptista, R., and Cilento, G. (1979) Photochem. Photobiol. 30, 101-110). In this paper many of the mechanistic details are established. The reaction is initiated by the autoxidation of IBAL to form the peracid (CH3)2CHC = O(OOH). The peracid converts horseradish peroxidase into compound I which in turn is converted into compound II by abstracting the alcoholic hydrogen atom from the enol form of IBAL. This creates a free radical with two resonance forms. (Formula: see text) Addition of molecular oxygen to the latter resonance form creates a peroxy radical which abstracts a hydrogen atom near the active site of the enzyme. The newly formed alpha-peroxide in turn forms a dioxetane-type of intermediate which rapidly decomposes into triplet acetone and formic acid. Compound II reacts with the enol by the same pathway as compound I. Thus native horseradish peroxidase is regenerated. The hydrogen atom abstraction near the enzyme active site may occur directly from ethanol, present to solubilize IBAL or from a group on the enzyme, in which case ethanol participates in a repair mechanism. Phosphate buffer is necessary because it catalyzes the keto-enol conversion of IBAL. Thus horseradish peroxidase participates in a normal peroxidatic cycle. The only chain reaction is the uncatalyzed autoxidation of IBAL, most of which occurs prior to the mixing of IBAL with the oxygenated horseradish peroxidase solution.     

Spectral properties of the higher oxidation states of prostaglandin H synthase

Prostaglandin H (PGH) synthase reacts with organic hydroperoxides and fatty acid hydroperoxides on a millisecond time scale to generate an intermediate that is spectrally similar to compound I of horseradish peroxidase. Compound I of PGH synthase is converted to compound II within 170 ms. Compound II decays to resting enzyme in a few seconds. Thus, the peroxidase reaction of PGH synthase appears to involve a cycle of native enzyme, compound I, and compound II, typical of heme-containing peroxidases. The Soret absorption maximum of compound I appears to occur at 412 nm but a small amount of compound II may be present. Soret maxima occur at 420, 433, and 419 for compound II, the ferrous enzyme, and the oxyferrous enzyme (compound III), respectively. Rapid scan analysis of the reaction of PGH synthase with arachidonic acid reveals the absorbance of compound II but no evidence for ferrous or oxyferrous enzyme.     

A kinetic and spectral study of the alkaline transitions of chloroperoxidase

The optical spectrum of chloroperoxidase in the near ultraviolet and visible region was studied from pH 6 to 12. Chloroperoxidase undergoes a first transition which is irreversible at pH 7 and a second transition near pH 11. The second transition is reversible provided the incubation period above pH 11 is kept as short as possible. The spectral properties of the intermediates were studied in the Soret region by means of a rapid scan apparatus. The rates of the transitions were measured in a stopped-flow apparatus. The pH dependence of both the spectra and the rate constants indicate that at least three ionizations are involved in the first alkaline transition.     

Similarities in the optical spectra of prostaglandin H synthase during its cyclooxygenase and peroxidase reactions

The spectral behavior of the enzyme prostaglandin H synthase was studied in the Soret region under conditions that permitted comparison of enzyme intermediates involved in peroxidase and cyclooxygenase activities. First, the peroxidase activity was examined. The enzyme's spectral behavior upon reacting with 5-phenyl-pent-4-enyl-1-hydroperoxide was different depending on the presence or absence of the reducing substrate, phenol. In the reaction of prostaglandin H synthase with the peroxide in the absence of phenol, formation of the enzyme intermediate compound I is observed followed by partial conversion to compound II and then by enzyme bleaching. In the reaction with both peroxide and phenol the absorbance decreases and a steady-state spectrum is observed which is a mixture of native enzyme and compound II. The steady state is followed by an increase in absorbance back to that of the native enzyme with no bleaching. The difference can be explained by the reactivity of phenol as a reducing substrate with the prostaglandin H synthase intermediate compounds. Cyclooxygenase activity with arachidonic acid could not be examined in the absence of diethyldithiocarbamate because extensive bleaching occurred. In the presence of diethyldithiocarbamate, enzyme spectral behavior similar to that seen in the reaction of the peroxide and phenol was observed. The similarity of the spectra strongly suggests that the enzyme intermediates involved in both the peroxidase and cyclooxygenase reactions are the same.     

Detection of ribosomal DNA sites in lentil and chickpea by fluorescent in situ hybridization.

Hybridization sites of an rDNA probe coding for the 18S, 5.8S, and 26S genes were detected on lentil and chickpea somatic chromosomes using fluorescent in situ hybridization. One pair of hybridization sites was detected in cultivated lentil Lens culinaris L. and wild lentil L. orientalis (Boiss.) Hand.-Mazz., and in both the hybridization sites of the ribosomal probe correspond to the secondary constriction. In cultivated chickpea Cicer arietinum three pairs of rDNA sites were detected and in the wild C. reticulatum two pairs were detected. The karyotypic relationship between the cultivated C. arietinum and its wild progenitor C. reticulatum is discussed.     

Reduction of prostaglandin H synthase compound II by phenol and hydroquinone, and the effect of indomethacin.

The reduction of prostaglandin H synthase compound II to native enzyme by phenol and by hydroquinone, in the presence of diethyldithiocarbamate as a stabilizing agent, was studied by rapid scan spectrometry and transient state kinetics at 4.0 +/- 0.5 degrees C in 0.1 M phosphate buffer, pH 8.0. The plot of pseudo-first-order rate constants for the conversion of prostaglandin H synthase compound II to native enzyme versus phenol concentration was linear with a non-zero intercept. The second-order rate constant was determined from the slope to be (5.3 +/- 0.3) x 10(5) M-1 s-1. For the reduction by hydroquinone, the second-order rate constant was determined from pointwise measurements of the pseudo-first-order rate constant to be (2.1 +/- 0.4) x 10(6) M-1 s-1. Rapid scan spectrum results also showed the reduction of compound I to compound II by both phenol and hydroquinone. Thus reduction of both compound I and compound II is one electron process. Our results suggest that the tyrosyl radical, detected in the presence of oxidizing agents, is formed by intramolecular electron transfer from the tyrosyl residue to the porphyrin pi-cation radical, and this reaction tends to disappear in the presence of sufficient reducing substrate. These in vitro results support speculation that there is a role of the peroxidase component of prostaglandin H synthase in benzene-induced toxicity. In the present work, the effect of indomethacin on the reduction of prostaglandin H synthase compound II by diethyldithiocarbamate, phenol, and hydroquinone was also investigated. Results revealed, for the first time, that indomethacin is an inhibitor of the peroxidase activity of prostaglandin H synthase, although not as effectively as in its well-known inhibition of cyclooxygenase activity.