The porphyrin-sensitized photooxidation of horseradish apoperoxidase.

Horseradish apoperoxidase (apoHRP) was reconstituted with various porphyrin derivatives, e.g., ferric, cupric, manganese, and zinc protoporphyrin IX, metal-free protoporphyrin IX, hematoporphyrin IX and deuteroporphyrin IX. The visible absorption spectra of these porphyrin-apoHRP complexes were examined. The time required for maximum development of the new Soret peak after reconstitution was used to measure the rate of porphyrin-apoHRP reconstitution. All of the four metal-protoporphyrins reconstituted with apoHRP at the same rate as metal-free protoporphyrin IX, whereas, for the metal-free porphyrins, the rates of reconstitution were in the order of deuteroporphyrin IX > hematoporphyrin IX > protoporphyrin IX. The porphyrins on the reconstituted porphyrin-apoHRP complexes were used as localized photosensitizers for photodynamic studies. No amino acid residues were oxidized on illumination of the ferric, cupric and manganese protoporphyrin IX-apoHRP complexes due to the paramagnetic properties of these metal ions. With diamagnetic zinc ion, two histidine and one methionine residues were oxidized which was the same as in the protoporphyrin IX- and hematoporphyrin IX-apoHRP complexes. However, only one histidine was destroyed on illumination of the deuteroporphyrin IX-apoHRP complex. The results confirmed the resistance of horseradish peroxidase to photodynamic action and suggested the involvement of at least one histidine residue in the heme environment of horseradish peroxidase.     

The hydrogen ion equilibria of horseradish peroxidase and apoperoxidase

1. The reversible proton dissociation equilibria of peroxidase, apoperoxidase and haem-recombined apoperoxidase have been explored in 150mm-potassium chloride at 20 degrees C at pH3-11.5. 2. Complementary heat measurements have been made of the classes of titratable groups to determine their intrinsic DeltaH dissociation. 3. These curves are interpreted as showing that there are two histidine residues capable of titration in peroxidase whereas there are three such in apoperoxidase. 4. Concomitant spectroscopic investigations indicate profound differences in the tyrosine ionizations in the two proteins. In peroxidase one group only of the five residues ionizes up to pH11.5. In apoperoxidase four residues are titratable. 5. Spectroscopic titration in 6m-guanidinium chloride and 150mm-potassium chloride reveal one tyrosine residue fewer in peroxidase than in apoperoxidase. 6. These findings are discussed in terms of the ;side chain' groups responsible for binding the haem group in peroxidase. A proximal imidazole group seems probable as is also the involvement of a distally placed tyrosine. 7. The differences between apo- and holo-peroxidase are stressed, particularly in respect of abnormal carboxyl group titration in the former.     

Sensitized photooxidation of low spin horseradish peroxidase.

Horseradish peroxidase differs from most enzymes in that it is almost completely resistant to photodynamic action due to the paramagnetic ferric ion in the prosthetic group, heme. Chelation of horseradish peroxidase at the sixth coordination position of the iron with a cyanide or hydroxyl group converts it to a low spin diamagnetic state. Upon illumination with visible light with eosin Y, flavin mononucleotide or methylene blue as sensitizer, the low spin enzyme lost both peroxidative and oxidative activities with the same quantum yields. Several amino acid residues, including one histidine and one tyrosine were destroyed in the low spin enzyme after 60 min of illumination with eosin Y as sensitizer.     

The course of photooxidation of menadione.

The highly purified (> 95%) mRNA coding for immunoglobulin light (L)-chain yields on acrylamide gels a discrete 15.5S band and a “shoulder” ranging in size from 15.5 to 9.5S. The “shoulder” was isolated and found to be fragmented mRNA as judged from: 1. hybridization kinetic analysis, using the complementary-DNA to the L-chain mRNA; 2. capacity to form aggregates, similarly to the intact 15.5S mRNA. Partial cleavage of the mRNA probably occurs during mechanical disruption of the myeloma cells. Fragments with intact 3′ end are selected due to binding via the poly(A) moiety to oligo(dT)-cellulose, i.e., the fragments should be deficient at the 5′ end where mRNA translation is initiated. In agreement, the fragmented mRNA is essentially untranslatable in a cell-free system. The size of the L-chain mRNA is rather uncertain. A value of 15.5S is obtained from migration in acrylamide gels made in water or formamide, a value of 12S is obtained from sucrose gradient centrifugation.     

Dye-sensitized photooxidation of phenanthrene

The dye-sensitized photooxidation of phenanthrene has been studied in a two-phase system employing $\text{n}$-hexane and water. Rose bengal was used as a sensitizer. A number of volatile oxidation products are observed and characterized by GC-MS-COM methods. The data suggest that one oxidation route involves the conversion of phenanthrene to 9,10-epoxy-9,10-dihydrophenanthrene which is related to the potentially carcinogenic arene oxides of more highly condensed polynuclear hydrocarbons. These results may have significance in connection with the enhancement of aberrant effects on biological systems produced by polynuclear hydrocarbons upon exposure to light.     

Structural dependence of oligonucleotide photooxidation

Oxidative photosensitization was used to characterize the conformational-dependent reactivity of various structures formed by oligonucleotides 14-15 nucleotides in length. The rate and product composition from a single hit process was analyzed using quantitative ion exchange chromatography under native and denaturing conditions. The primary damage incurred under aerobic acetone sensitization was base oxidation that, in turn, would induce strand scission upon a secondary treatment with piperidine. The reactive intermediates of this process were not consistent with diffusible radical species or singlet oxygen, as indicated by isotope and quenching studies. Derivatization was most likely initiated through a type I photoprocess with a direct interaction between DNA bases and excited state acetone preceding an irreversible oxidation step. This dominant reaction demonstrated no obvious sequence or site specificity for initial modification; the relative reactivity among the oligonucleotides did not correspond to any simple trend of base composition or near neighbor analysis. Likewise, the steric requirements of base modification allowed for similar rates of oxidation for single-strand, helical, and aberrant forms of DNA. Hybridization of the most reactive oligonucleotides, however, did suppress their relative single-strand vs double-strand reactivity by as much as fourfold.     

The reaction of coumarins with horseradish peroxidase

The peroxidase catalyzed oxidation of indole-3-acetate is inhibited by naturally occurring coumarins such as scopoletin. This inhibition is due to the preferential reactivity of the coumarins with the peroxidase compounds I, II, and III. In view of the possible growth regulatory role of coumarins in plants, the mechanism of oxidation of scopoletin by horse-radish peroxidase has been investigated.     

The permeability of muscle capillaries to horseradish peroxidase

The main objective of this study was to determine the pathways by which horseradish peroxidase (HRP) can cross the endothelium of muscle capillaries. Specimens of mouse diaphragm were fixed for cytochemical analysis at various intervals after intervenous injection of 0.5 mg HRP, at 4 min after intervenous injection of varied amounts of HRP, and at 4 min after intervenous injections in various volumes of isotonic NaCl. Our findings indicate that endothelial junctions serve as a barrier which may allow passage of very limited amounts of HRP. They also suggest that endothelial vesicles transfer HRP from the capillary lumen to the pericapillary interstitium as well as in the reverse direction. Increasing the volume of solution injected to approximately 30% of total blood volume did not increase the amount of HRP that left the capillary lumen. Our results with HRP do not provide clearcut evidence that endothelial junctions are the site of the small pore.     

Studies on the photooxidation of tryptophan

1.

1. From the study of the photooxidation of tryptophan in three different aqueous systems, it has been concluded that the primary reaction is one which leads to the formation of formylkynurenine. Secondary reactions result in the formation of a variety of simpler amino acids together with a complex mixture of aromatic and polymeric materials.     

The cDNA sequence of a neutral horseradish peroxidase

A cDNA clone encoding a horseradish (Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic pI value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition with the HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.     

Carotenoid photooxidation in photosystem II

Carotenoids are known to function as light-harvesting pigments and they play important roles in photoprotection in both plant and bacterial photosynthesis. These functions are also important for carotenoids in photosystem II. In addition, beta-carotene recently has been found to function as a redox intermediate in an alternate pathway of electron transfer within photosystem II. This redox role of a carotenoid in photosystem II is unique among photosynthetic reaction centers and stems from the very highly oxidizing intermediates that form in the process of water oxidation. In this minireview, an overview of the electron-transfer reactions in photosystem II is presented, with an emphasis on those involving carotenoids. The carotenoid composition of photosystem II and the physical methods used to study the structure of the redox-active carotenoid are reviewed. Possible roles of carotenoid cations in photoprotection of photosystem II are discussed.