Superoxide ion as a primary reductant in ascorbate-mediated ferritin iron release.

The mechanism of ascorbate-promoted ferritin iron reduction under aerobic conditions was studied. The initial rate of ferritin iron release was determined by spectrophotometric measurement of the Fe(ferrozine)3(2+) complex which absorbs at 562 nm. Variation of the initial ferrozine concentration had no influence on the rate of iron release suggesting that ferrozine does not participate in the rate-determining step. Experimental measurements of the initial rate of iron release as a function of ascorbate concentration resulted in saturation kinetics with Vmax = 2.0 X 10(-7) M.min-1 and KM = 1.3 X 10(-3) M. The effect of pH was quite pronounced with a maximal rate of iron release at pH 7.0. Stoichiometric measurements on the reaction mixture, with added catalase, resulted in a ratio of 2 Fe(II) released per ascorbate. Ascorbate-mediated iron release was inhibited 85% by superoxide dismutase, but 0% inhibition was noted with aposuperoxide dismutase. It is proposed that superoxide ion, generated during the iron-promoted oxidation of ascorbate, acts as a reductant of ferritin iron. A mechanism of ferritin iron release consistent with these experimental observations is discussed.

The action of lipoxygenase-1 on furan derivatives.

Several 2,5-disubstituted furans, which are known to react with peroxyacids, singlet oxygen and other active forms of oxygen were tested as potential inhibitors, co-oxidants, or substrates for soybean lipoxygenase. The furan, 10,13-epoxy-octadeca-10,12-dienoic acid, methyl ester (IV) was converted by lipoxygenase or singlet oxygen or peroxyacid to the acyclic product, methyl 10,13-dioxo-octadec-11-enoate. Apparently furan IV is able to interact with an active site of lipoxygenase (Km = 220 microM). 2,5-Dimethylfuran (I), 2,5-diphenylfuran (II) and 3-(5'-methyl-2'-furyl)propenoic acid (III) were neither substrates nor inhibitors of lipoxygenase activity. Lipoxygenase-catalyzed oxidation of furan (IV), which is inhibited by hydroquinone, is explained by a mechanism involving lipoxygenase-superoxide complex and furan-radical intermediates. Also described is the selective cleavage of furan rings by m-chloroperoxybenzoic acid to yield the 1,4-diketoethylene functional system.

Reduction and release of ferritin iron by plant phenolics.

The reductive release of ferritin iron by several naturally occurring o-diphenols was studied. The initial rate of iron release was quantified by spectrophotometric measurement of the Fe(ferrozine)3(2+) complex, which absorbs maximally at 562 nm. The initial rate of iron release was dependent upon o-diphenol concentration, but not on the concentration of the chromophoric chelating agent, ferrozine, Stoichiometric measurements resulted in a ratio of 2Fe(II) released per molecule of o-diphenol. The series of o-diphenols studied included, caffeic acid, chlorogenic acid, dihydrocaffeic acid, 3,4-dihydroxybenzoic acid, and several analogs. These reductants represent an oxidation reduction potential range of 0.38 volts. A direct correlation between reducing power of the o-diphenols and rate of ferritin iron release was observed. Superoxide dismutase, catalase, mannitol, or general radical traps had no effect on the rate of iron removal; however, EDTA and oxalate inhibited iron release. A mechanism for ferritin iron reduction and release by o-diphenols consistent with the experimental observations is discussed.

Iron incorporation into apoferritin. The role of apoferritin as a ferroxidase.

Apoferritin catalyzes the oxidation of Fe(II) to Fe(III). Ferroxidase activity is assayed and characterized by coupling the oxidation with the binding of Fe(III) to transferrin. The initial rate of Fe(II) oxidation is dependent on apoferritin and initial Fe(II) concentration but independent of transferrin concentration. The ferroxidase activity is inhibited by Zn(II). Ferritins with varying loads of iron have the same ferroxidase activity level. It is suggested that the described oxidation process represents the initial step of iron deposition in apoferritin. Since transferrin can intercept Fe(III) before it is deposited in apoferritin, active sites for Fe(II) oxidation must be on or near the surface of apoferritin. This finding is contrary to the current view of apoferritin-catalyzed oxidation of Fe(II) which places active sites in the channels to the core or inside the central core.

The incorporation of iron into apoferritin as mediated by ceruloplasmin.

Ceruloplasmin, a copper ferroxidase, promotes the incorporation of Fe(III) into the iron storage protein, apoferritin. The product formed is identical to ferritin as judged by polyacrylamide electrophoresis and iron/protein measurements. Of several proteins examined, only apoferritin accumulates the Fe(III) produced by ceruloplasmin. When ceruloplasmin was replaced by tyrosinase, which we have shown to have ferroxidase activity, no iron incorporation into apoferritin was observed. It is proposed that Fe(III) is transferred directly and specifically to apoferritin. These data support a more specific role for ceruloplasmin in iron metabolism than has previously been proposed.

Effect of chemical modification of cellulose on the activity of a cellulase from Aspergillus niger.

Five chemically modified forms of cellulose were prepared, characterized, and tested as substrates for a homogeneous glucanohydrolase from A. niger. The relative order of reactivity at pH 4.0 was DEAE = PEI > benzyl DEAE > cellulose > P > CM.The following abbreviations are used throughout the article: (RBB) Remazol brilliant blue R; (DEAE) diethylamino ethyl; (PEI) polethyleneimine; (CM) carboxymenthyl; (P) phospho; (DS) degree of RBB dye substitution of cellulose, in mol dye/100 glucose. This indicates that positively charged cellulose substrates are more susceptible to hydrolysis by the cellulase. This observation strengthens an earlier proposal that caroxyl groups on the enzyme are involved in substrate binding and catalytic action. Chemical modification is suggested as a method to increase the rate of enzymatic hydrolysis of cellulose, a process now in the commercial development stage.

Reductive release of ferritin iron: a kinetic assay.

Ferritin iron release, a process of considerable interest in biology and medicine, occurs most readily in the presence of reducing agents. Here is described a kinetic assay for measuring the rate of ferritin iron removal promoted by various reductants. The new procedure uses ferrozine as a chromophoric, high-affinity chelator for the product, Fe(II). The initial rate of iron release is quantified by continuous spectrophotometric measurement of the Fe(ferrozine)2/3+ complex which absorbs maximally at 562 nm. The initial rate of iron mobilization is dependent on reductant concentration, but not on the concentration of the chelating agent, ferrozine. Saturation kinetics are observed for all reductants, including dihydroxyfumarate, cysteine, caffeic acid, ascorbate, and glutathione. Superoxide dismutase greatly inhibits ferritin iron release by ascorbate, but has little or no effect on the reducing action of dihydroxyfumarate, cysteine, caffeic acid, or glutathione. Ferritin iron removal by dihydroxyfumarate was inhibited by various metal ions. This new assay may be used for rapid screening of test compounds for treatment of iron overload and for investigation of the mechanistic aspects of ferritin iron reduction.

Fractionation of Aspergillus niger cellulases by combined ion exchange affinity chromatography.

Eight chemically modified cellulose supports were tested for their ability to absorb components of the Aspergillus niger cellulase system. At least two of the most effective adsorbents, aminoethyl cellulose and carboxymethyl cellulose, were shown to be useful for the fractionation of cellulases. These supports apparently owe their resolving capacity to both ion exchange and biospecific binding effects; however, the relative importance of each effect is unknown. These observations form the basis for a new cellulase fractionation technique, combined ion exchange-affinity chromatography.

Selective adsorption of apoferritin on immobilized Fe(III): demonstration of Fe(III) binding sites.

Immobilized metal ion affinity chromatography has been used to demonstrate and partially characterize Fe(III) binding sites on apoferritin. Binding of Fe(III) to these sites is influenced by pH, but not affected by high ionic strength. These results suggest that both ionic and coordinate covalent interactions are important in the formation of the Fe(III): apoferritin complex. This is, to our knowledge, the first demonstration of direct Fe(III) binding to apoferritin. Other immobilized metal ions, including Zn(II), Ni(II), Cu(II), Cr(III), Co(II), and Tb(III), displayed little or no adsorption of apoferritin. The analytical technique of immobilized metal ion affinity chromatography also shows great promise in the purification of apoferritin, ferritin, and other iron-binding proteins.