ABSTRACT
Ferrochelatase (FECH), the enzyme at the last step of the heme-biosynthetic pathway, is involved in the formation of Zn-protoporphyrin via an iron-removal reaction of heme. To improve the efficacy of the formation of Zn-protoporphyrin from heme, the use of recombinant FECHs from porcine, yeast, and bacteria was examined. Incubation of FECH with myoglobin in the presence of ascorbic acid and cysteine resulted in the efficient conversion of myoglobin-heme to Zn-protoporphyrin. Exogenously added recombinant yeast FECH facilitates the production of Zn-protoporphyrin from myoglobin-heme and heme in meat, via the replacement of iron in the protoporphyrin ring by zinc ions. A large amount of Zn-protoporphyrin was also generated by the catalysis of FECH using an intact piece of meat as a substrate. These findings can open up possible approaches for the generation of a nontoxic bright pigment, Zn-protoporphyrin, to shorten the incubation time required to produce dry-cured ham.
Subject(s)
Bacterial Proteins/chemistry , Ferrochelatase/chemistry , Heme/chemistry , Meat Products/analysis , Protoporphyrins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Zinc/chemistry , Animals , Ascorbic Acid/chemistry , Bacterial Proteins/genetics , Catalysis , Ferrochelatase/genetics , Food Preservation , Iron/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomycetales/enzymology , Saccharomycetales/genetics , Swine , Thermus thermophilus/enzymology , Thermus thermophilus/geneticsABSTRACT
Exogenous δ-aminolevulinic acid (ALA)-induced photodynamic therapy (PDT) has been used in the treatment of cancer. To obtain a high efficacy of ALA-PDT, we have screened various chemicals affecting ALA-induced accumulation of protoporphyrin in cancerous cells. When HeLa cells were treated with quinolone chemicals including enoxacin, ciprofloxacin or norfloxacin, the ALA-induced photodamage accompanied by the accumulation of protoporphyrin was stronger than that with ALA alone. Thus, quinolone compounds such as enoxacin, ciprofloxacin and norfloxacin enhanced ALA-induced photodamage. The increased ALA-induced photodamage in enoxacin-treated HeLa cells was decreased by haemin or ferric-nitrilotriacetate (Fe-NTA), suggesting that an increase in iron supply cancels the accumulation of protoporphyrin. On the other hand, the treatment of the cells with ALA plus an inhibitor of haem oxygenase, Sn-protoporphyrin, led to an increase in the photodamage and the accumulation of protoporphyrin compared with those upon treatment with ALA alone, indicating that the cessation of recycling of iron from haem augments the accumulation. The use of quinolones plus Sn-protoporphyrin strongly enhances ALA-induced photodamage. To examine the mechanisms involved in the increased accumulation of protoporphyrin, we incubated ferric chloride with an equivalent amount of quinolones. Iron-quinolone complexes with visible colours with a maximum at 450 nm were formed. The levels of iron-metabolizing proteins in enoxacin- or ciprofloxacin-treated cells changed, indicating that quinolones decrease iron utilization for haem biosynthesis. Hence, we now propose that the use of quinolones in combination with ALA may be an extremely effective approach for the treatment modalities for PDT of various tumour tissues in clinical practice.
Subject(s)
Aminolevulinic Acid/pharmacology , Fluoroquinolones/pharmacology , Photochemotherapy/methods , Photosensitizing Agents/pharmacology , Protoporphyrins/metabolism , Aminolevulinic Acid/chemistry , Drug Synergism , Female , Ferric Compounds/metabolism , Fluoroquinolones/chemistry , HeLa Cells , Heme Oxygenase (Decyclizing)/antagonists & inhibitors , Heme Oxygenase (Decyclizing)/metabolism , Hemin/metabolism , Humans , Iron/metabolism , Light , Metalloporphyrins/pharmacology , Nitrilotriacetic Acid/analogs & derivatives , Nitrilotriacetic Acid/metabolism , Oxidation-Reduction , Photosensitizing Agents/chemistry , Protoporphyrins/pharmacology , Reactive Oxygen Species/metabolism , Skin Neoplasms/metabolism , Skin Neoplasms/therapyABSTRACT
At the terminal step of heme biosynthesis, ferrochelatase (FECH) catalyzes the insertion of Fe2+ into protoporphyrin to form heme. It is located on the inner membrane of the mitochondria of animals. The enzyme inserts divalent metal ions, including Fe2+, Co2+, and Zn2+, into porphyrins in vitro. We have reported that it can remove Fe2+ from heme. To characterize the iron-removal reverse activity of FECH, we examined its properties in porcine liver and muscle mitochondria, and isolated porcine FECH cDNA. The amino acid sequence of porcine FECH showed high homology with bovine (91%), human (85%), mouse (87%), and rat (76%) equivalents. It was expressed in Escherichia coli, and purified, and the kinetic properties of the zinc-chelating and iron-removal activities were examined. Both activities peaked at 45 degrees C, but different optimal pH values, of 7.5-8.0 for zinc-ion insertion and 5.5-6.0 for the reverse reaction were found. The K(m) values for mesoporphyrin IX and Zn2+ were 6.6 and 1.1 microM, respectively, and the K(m) for heme was 5.7 microM. The k(cat) value of the forward reaction was about 11-fold higher than that of the reverse reaction, indicating that the enzyme preferably catalyzes the forward reaction rather than the iron-removal reaction. Reverse activity was stimulated by fatty acids and phospholipids, similarly to the case of the forward reaction, indicating that lipids play a role in regulating both enzyme activities.
