CHEMIENERGIZED SPECIES IN PEROXIDASE SYSTEMS

Abstract— Several hemeprotein-catalyzed reactions generate products of the type expected from the cleavage of a high energy intermediate. For some systems, the formation, in high yield, of a carbonyl compound in its excited triplet state has been firmly established on the basis of (i) equivalence of the chemiluminescence and phosphorescence spectra of the expected products; (ii) energy transfer to sensitizers containing heavy atoms and (iii) occurrence of photoproducts. The excited species appears to be generated within the enzyme and shielded from quenching by oxygen. It may be quenched, however, via long-range triplet-singlet energy transfer.
This work strongly supports our hypothesis that excited electronic states are also formed in biological systems which are not necessarily bioluminescent. One of the functions which peroxidases may thus fulfill might be the utilization of the potential of photochemistry in the absence of light.     

CONJUGATED DIENE FORMATION PROMOTED BY TRIPLET ACETONE ACTING UPON ARACHIDONIC ACID

Abstract— Post UV-B(280–320 nm) exposure to UV-A(320–400 nm) reverses pyrimidine dimers in the epidermal DNA of the South American opossum Monodelphis domestica [Ley, R. D. (1984) photorepair of pyrimidine dimers in the epidermis of the marsupial Monodelphis domestica. Photochem. Photobiol . 40 ,141–143.] To demonstrate that the observed photorepair is mediated by an enzyme, we have isolated a DNA photolyase from the opossum. DNA photolyase from liver was purified 3000-fold by ammonium sulfate fractionation and phenylsepharose, hydroxylapatite, DEAE-cellulose and DNA-cellulose column chromatography. Heat denaturation (60°C for 4 min) completely eliminated the photoreactivating activity. The enzyme was active in the pH range of 5.5 to 8.5 with a pH optimum of 7.5. The enzyme has an apparent molecular weight of 32 000 under nondenaturing conditions. The activity of the enzyme was not affected by sodium chloride up to 250 m M . The action spectrum for the purified DNA photolyase showed activity in the range of325–475 nm with peak actvity at 375 nm.     

Tris(Bipyridine) Ruthenium(II): An Efficient Detector of Excited Species Generated by Chemiluminescent Processes*

In systems that are known to generate electronically excited species, the tris(bipyridine) ruthenium(II) cation, Ru(bpy)₃²⁺, ion is found to be an efficient, chemically stable, emissive energy acceptor for probing excited state formation. The chemiexcitation system employed was the thermolysis of tetramethyldioxetane. The system employed for enzymatic generation of excited species was the horseradish peroxidase-catalyzed oxidation of appropriate substrates such as isobutyraldehyde, phenylacetal-dehyde and indoleacetic acid. The versatility of the Ru(bpy)₃²⁺ ion is exemplified by its capacity to detect excited state formation in both chemical and enzymatic systems and in homogeneous and microheterogeneous media. The long wavelength emission and chemical stability of Ru(bpy)₃²⁺ facilitate quantitative studies of the sensi-tization process. Possible routes leading to excitation of the Ru(bpy)₃²⁺ ion by the chemi- or enzyme-generated excited species include electronic energy transfer and electron transfer-induced luminescence.     

GENERATION OF ELECTRONICALLY EXCITED STATES IN SITU. POLYMORPHONUCLEAR LEUKOCYTES TREATED WITH PHENYLACETALDEHYDE

In polymorphonuclear leukocytes phenylacetaldehyde promotes an intracellular O2 consuming process in which myeloperoxidase participates. The reaction is accompanied by lipid peroxidation as shown by both malondialdehyde formation and biphasic light emission. The lipid peroxidation appears to be induced by intracellularly generated triplet benzaldehyde. When chlorophyll-a is solubilized in the leukocytes, biphasic emission is observed in the red, demonstrating that the excited species formed in lipid peroxidation transfer their energy to chlorophylls bound to the cell. The energy transfer process is efficient and does not occur by radiative transfer.     

ON THE POSSIBILITY OF GENERATION AND TRANSFER OF ELECTRONIC ENERGY IN BIOCHEMICAL SYSTEMS

Abstract— Generation of electronically excited species is a distinct possibility in biological systems; the most likely ones at present appear to be flavins, metalloporphyrins, oxygen and certain conjugated carbonyl compounds. Thyroxine should be very active in certain cases of electronic energy transfer.     

DUAL EFFECTS OF TRYPTOPHAN IN THE HORSERADISH PEROXIDASE SYSTEM THAT GENERATES TRIPLET ACETONE

When tryptophan is added to the horseradish peroxidase/hydrogen peroxide system, it markedly increases the rate of 'spontaneous' conversion of peroxidase-compound II to the native form without undergoing any alteration by spectral criteria. The D-isomer is more efficient than the L-isomer. The latter binds to the enzyme compound II, although at a site not very favourable for reduction. The accelerating effect of tryptophan is also seen when isobutyraldehyde or its enol added to the system, is converted into triplet acetone. Again the D-isomer is more efficient than the L-isomer. The D-isomer is also more efficient in quenching the acetone phosphorescence. These results indicate that, at least in the case of L-tryptophan where complexation with compound II occurs, the interaction of tryptophan with triplet acetone bound to the enzyme takes place in a ternary complex.