Iron is the intracellular metal involved in the production of DNA damage by oxygen radicals.

The metal chelators 1,10-phenanthroline and 2,9-dimethyl-1,10-phenanthroline (neocuproine) showed distinct abilities to prevent hydroxyl radical formation from hydrogen peroxide and Cu+ or F2(2+) (Fenton reaction) as determined by electron spin resonance. o-Phenanthroline prevented both Fe- and Cu-mediated Fenton reactions whereas neocuproine only prevented the Cu-mediated Fenton reaction. Because only 1,10-phenanthroline but not neocuproine prevented DNA strand-break formation in hydrogen peroxide-treated mammalian fibroblasts it appears that the Fe-mediated, as compared to the Cu-mediated, intranuclear Fenton reaction is responsible for DNA damage.     

A comparison of cobalt(II) and iron(II) hydroxyl and superoxide free radical formation

We have employed the electron spin resonance spin-trapping technique to study the reaction of Co(II) with hydrogen peroxide in a chemical system and in a microsomal system. In both cases, we employed the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and were able to detect the formation of DMPO/.OH and DMPO/.OOH. DMPO/.OOH was the predominant radical adduct formed in the chemical system, while the two adducts were of similar concentrations in the microsomal system. The formation of both of these adducts in either reaction system was inhibited by the addition of superoxide dismutase or catalase, and by chelating the cobalt with either ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). The incorporation of the hydroxyl radical scavengers ethanol, formate, benzoate, or mannitol inhibited the formation of DMPO/.OH in both systems. We also repeated the study using Fe(II) in place of Co(II). In contrast to the Co(II) results, Fe(II) reacted with hydrogen peroxide to yield only DMPO/.OH, and this adduct formation was relatively insensitive to the presence of added superoxide dismutase. In addition, Fe(II)-mediated DMPO/.OH formation increased when the iron was chelated to either EDTA or DTPA rather than being inhibited as for Co(II). Thus, we propose that Co(II) does not react with hydrogen peroxide by the classical Fenton reaction at physiological pH values.     

Redox regulation of proliferation of lens epithelial cells in culture

Both oxidants and antioxidants have been shown to modulate cell proliferation. We studied the effects of hydrogen peroxide and two antioxidants on the rate of proliferation of lens epithelial cells in culture. Hydrogen peroxide at concentrations higher than 32 microM caused a significant inhibition of proliferation. However, in the concentration range of 0.01-0.5 microM, hydrogen peroxide stimulated the rate of proliferation. The effect of hydrogen peroxide was dependent on the amount of cells in an individual culture well, indicating decomposition of hydrogen peroxide by cellular enzymes. In order to eliminate the possibility of decomposition of the dose of hydrogen peroxide given as a bolus, we induced continual production of hydrogen peroxide by adding glucose oxidase to the incubation medium. We found that hydrogen peroxide, generated by 1-50 microU x ml(-1) of glucose oxidase significantly increased the rate of cell proliferation. This effect was most apparent at the beginning of the exponential phase of cellular growth. Glucose oxidase alone (100-500 microU x ml(-1)) did not produce any effect. The effects of pro-oxidative hydrogen peroxide were compared with the effects of two biologically important antioxidants, alpha-tocopherol and retinol. Both antioxidants completely inhibited proliferation at concentrations of 30 microM and higher. In contrast to retinol, the effect of alpha-tocopherol was dependent on the amount of cells, indicating cellular decomposition of alpha-tocopherol. The results document the possibility of redox regulation of cellular proliferation at physiologically relevant reactant concentrations.     

Cleavage of recombinant proteins at poly-His sequences by Co(II) and Cu(II)

Improved ways to cleave peptide chains at engineered sites easily and specifically would form useful tools for biochemical research. Uses of such methods include the activation or inactivation of enzymes or the removal of tags for enhancement of recombinant protein expression or tags used for purification of recombinant proteins. In this work we show by gel electrophoresis and mass spectroscopy that salts of Co(II) and Cu(II) can be used to cleave fusion proteins specifically at sites where sequences of His residues have been introduced by protein engineering. The His residues could be either consecutive or spaced with other amino acids in between. The cleavage reaction required the presence of low concentrations of ascorbate and in the case of Cu(II) also hydrogen peroxide. The amount of metal ions required for cleavage was very low; in the case of Cu(II) only one to two molar equivalents of Cu(II) to protein was required. In the case of Co(II), 10 molar equivalents gave optimal cleavage. The reaction occurred within minutes, at a wide pH range, and efficiently at temperatures ranging from 0 degrees C to 70 degrees C. The work described here can also have implications for understanding protein stability in vitro and in vivo.     

The hydrogen peroxide reactivity of peptidylglycine monooxygenase supports a Cu(II)-superoxo catalytic intermediate

We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H(2)O(2), the alpha-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When (18)O-labeled H(2)O(2) was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with (18)O and must therefore be derived from H(2)O(2). However, when the reaction was carried out with H (16)(2)O(2) in the presence of (18)O(2), 60% of the product contained the (18)O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the Cu(H) center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.     

Chemiluminescent determination of copper in the serum

A procedure for chemiluminescent determination of copper in the blood serum ash is suggested using an indicator reaction between 4-aminophthalhydrazide and hydrogen peroxide permitting copper to be determined in a minimum (0.003 micrograms) concentrations in 1 ml of the blood serum.     

Iodide oxidation and iodine reduction mediated by horseradish peroxidase in the presence of ethylenediaminetetraacetic acid (EDTA): the superoxide effect

Ethylenediaminetetraacetic acid (EDTA) is an inhibitor of iodide (I-) oxidation that is catalyzed by horseradish peroxidase (HRP). HRP-mediated iodine (I2) reduction and triiodide (I3+) disappearance occur in the presence of this inhibitor. It is interesting that in the presence of EDTA, HRP produces superoxide radical, a reactive oxygen species that is required for iodine reduction. Substitution of potassium superoxide (KO2) or a biochemical superoxide generating system (xanthine/xanthine oxidase) for HRP and H2O2 in the reaction mixture also can reduce iodine to iodide. Thus, iodine reduction mediated by HRP occurs because HRP is able to mediate the formation of superoxide in the presence of EDTA and H2O2. Although superoxide is able to mediate iodine reduction directly, other competing reactions appear to be more important. For example, high concentrations (mM range) of EDTA are required for efficient iodine reduction in this system. Under such conditions, the concentration (microM range) of contaminating EDTA-Fe(III) becomes catalytically important. In the presence of superoxide, EDTA-Fe(III) is reduced to EDTA-Fe(II), which is able to reduce iodine and form triiodide rapidly. Also of importance is the fact that EDTA-Fe(II) reacts with hydrogen peroxide to form hydroxyl radical. Hydroxyl radical involvement is supported by the fact that a wide variety of hydroxyl radical (OH) scavengers can inhibit HRP dependent iodine reduction in the presence of EDTA and hydrogen peroxide.     

Interactions of quercetin with iron and copper ions: complexation and autoxidation

Quercetin (3,3',4',5,7-pentahydroxyflavone), one of the most abundant dietary flavonoids, has been investigated for its ability to bind Fe(II), Fe(III), Cu(I) and Cu(II) in acidic to neutral solutions. In particular, analysis by UV-visible spectroscopy allows to determine the rate constants for the formation of the 1:1 complexes. In absence of added metal ion, quercetin undergoes a slow autoxidation in neutral solution with production of low hydrogen peroxide (H(2)O(2)) concentrations. Autoxidation is accelerated by addition of the metal ions according to: Cu(I) > Cu(II)>Fe(II) Fe(III). In fact, the iron-quercetin complexes seem less prone to autoxidation than free quercetin in agreement with the observation that EDTA addition, while totally preventing iron-quercetin binding, slightly accelerates quercetin autoxidation. By contrast, the copper-quercetin complexes appear as reactive intermediates in the copper-initiated autoxidation of quercetin. In presence of the iron ions, only low concentrations of H(2)O(2) can be detected. By contrast, in the presence of the copper ions, H(2)O(2) is rapidly accumulated. Whereas Fe(II) is rapidly autoxidized to Fe(III) in the presence or absence of quercetin, Cu(I) bound to quercetin or its oxidation products does not undergo significant autoxidation. In addition, Cu(II) is rapidly reduced by quercetin. By HPLC-MS analysis, the main autoxidation products of quercetin are shown to be the solvent adducts on the p-quinonemethide intermediate formed upon two-electron oxidation of quercetin. Finally, in strongly acidic conditions (pH 1-2), neither autoxidation nor metal complexation is observed but Fe(III) appears to be reactive enough to quickly oxidize quercetin (without dioxygen consumption). Up to ca. 7 Fe(III) ions can be reduced per quercetin molecule, which points to an extensive oxidative degradation.     

Characterization of reactions catalyzed by manganese peroxidase from Phanerochaete chrysosporium

Manganese peroxidase (MnP) is one of two extracellular peroxidases believed to be involved in lignin biodegradation by the white-rot basidiomycete Phanerochaete chrysosporium. The enzyme oxidizes Mn(II) to Mn(III), which accumulates in the presence of Mn(III) stabilizing ligands. The Mn(III) complex in turn can oxidize a variety of organic substrates. The stoichiometry of Mn(III) complex formed per hydrogen peroxide consumed approaches 2:1 as enzyme concentration increases at a fixed concentration of peroxide or as peroxide concentration decreases at a fixed enzyme concentration. Reduced stoichiometry below 2:1 is shown to be due to Mn(III) complex decomposition by hydrogen peroxide. Reaction of Mn(III) with peroxide is catalyzed by Cu(II), which explains an apparent inhibition of MnP by Cu(II). The net decomposition of hydrogen peroxide to form molecular oxygen also appears to be the only observable reaction in buffers that do not serve as Mn(III) stabilizing ligands. The nonproductive decomposition of both Mn(III) and peroxide is an important finding with implications for proposed in vitro uses of the enzyme and for its role in lignin degradation. Steady-state kinetics of Mn(III) tartrate and Mn(III) malate formation by the enzyme are also described in this paper, with results largely corroborating earlier findings by others. Based on a comparison of pH effects on the kinetics of enzymatic Mn(III) tartrate and Mn(III) malate formation, it appears that pH effects are not due to ionizations of the Mn(III) complexing ligand.     

The effects of chelating agents on radical generation in alkaline peroxide systems, and the relevance to substrate damage

A spin-trapping EPR technique has been employed to explore the generation of hydroxyl radicals from reactions between a series of first row transition metal ions and aqueous hydrogen peroxide at pH 10, and with a range of chelating agents (EDTA, DTPMP and the readily biodegradable ligands S,S-EDDS and IDS). In the absence of these chelating agents only Cu(II) generates a significant level of hydroxyl radicals; in their presence with Cu(II) EDTA and IDS give similar behaviour whereas EDDS and DTPMP inhibit hydroxyl radical generation. For Fe(II), EDTA, DTPMP and IDS significantly enhance ^√OH production under these conditions whereas EDDS does not. Results from model cellulose damage experiments broadly confirm the findings for copper, though experiments with Fe(II) lead to somewhat contrasting results. Our findings are discussed in terms of binding constants and implications for alkaline peroxygen bleaching systems.     

γ-Butyrobetaine hydroxylase: A unique protective effect of catalase

Catalase stimulates the activity of homogeneous γ-butyrobetaine hydroxylase by approximately 300-fold. The stimulation of the hydroxylation reaction elicited by catalase is saturable, and although a number of proteins may be substituted for catalase, none is as effective. γ-Butyrobetaine hydroxylase is also irreversibly inactivated in the presence of one of its substrates, oxygen, and its cofactor, ascorbate. This inactivation of the hydroxylase activity may be prevented by (i) the presence of high concentrations (2 mg/ml) of various proteins, (ii) the presence of catalytic concentrations (20 μg/ml) of catalase, or (iii) the presence of 10 mm histidine or dithiothreitol. Oxidized species of ascorbate do not appear to be responsible for the inactivation process. Time-dependent inactivation is also observed when γ-butyrobetaine hydroxylase is preincubated with hydrogen peroxide generated by the glucose oxidase-catalyzed oxidation of glucose. At low concentrations, superoxide dismutase was not as effective as an equivalent protein concentration of catalase in protecting against inactivation, and hydroxyl radical scavengers were completely ineffective. In measurements of γ-butyrobetaine hydroxylase activity, the presence of catalase both stimulates the catalytic activity of the hydroxylase and protects the enzyme from inactivation by a product of the interaction of components in the assay mixture, presumably hydrogen peroxide.     

Peroxidase and hydrogen peroxide detection by a bioenergized method

Peroxidase activity and hydrogen peroxide concentrations were measured by a chemiluminescent method based upon the reduction of peroxidase Compounds I and II by both eosin and EDTA. Eosin excess present in the reaction mixture was excited during the reaction to its fluorescent state. This bioenergized method allows calculation of peroxidase concentration in the range of 10^?12 to 10^?7m, and hydrogen peroxide concentration in the range of 10^?9 to 10^?5m. This approach has been applied to the estimation of peroxidase activity in human red cell membranes and hydrogen peroxide formation in the peroxidase-catalyzed oxidation of glutathione.     

Effect of lipophilic chelators on oxyradical-induced DNA strand breaks in human granulocytes: paradoxical effect of 1,10-phenanthroline.

Strand breaks can be produced in the DNA of intact granulocytes by a flux of oxyradicals (O2- and H2O2) generated by tetradecanoylphorbol acetate (TPA) or by a flux of H2O2 generated by glucose oxidase. The mechanism by which such breaks are induced is still uncertain. Lipophilic chelators such as dipyridyl and 1,10-phenanthroline (OP) strongly inhibit strand breaks induced by H2O2, presumably because of their ability to chelate intracellular iron. We now report that dipyridyl also partially inhibits strand breaks in TPA-stimulated granulocytes while a "copper-specific" lipophilic chelator, neocuproine, has no effect. As opposed to these effects, OP increases the number of strand breaks in TPA-stimulated granulocytes. Superoxide dismutase (SOD) (but not catalase) partially blocks this increase. Both the cell-impermeable chelator, EDTA, and neocuproine strongly block the increase also. In fact, in the presence of EDTA, OP behaves like dipyridyl and inhibits strand breaks. Preformed OP2-copper(II) complex causes DNA breaks in TPA-stimulated granulocytes. The paradoxical effect of OP may be explained by assuming that OP may form two different metal complexes, a DNA-damaging complex with copper or an inhibitory complex with iron. If copper(II) and O2- are present, the first complex may form and the net effect may be an increase in strand breaks. If the formation of this complex is prevented by SOD, EDTA, or neocuproine, then OP may complex iron and the net effect may be (like dipyridyl) an inhibition of strand breaks. The source of the copper responsible for the formation of OP2-copper complex is unknown.     

Activation of lecithin-cholesterol acyltransferase by apolipoprotein D: comparison of proteoliposomes containing apolipoprotein D, A-I or C-I

To study the activation of lecithin-cholesterol acyl transferase (LCAT) (phosphatidylcholine:sterol O-acyltransferase, EC 2.3.1.43) by apolipoprotein D in comparison to apolipoproteins A-I and C-I, proteoliposomes with a phosphatidylcholine/free cholesterol molar ratio of 24:1, containing 10-300 micrograms/ml of apolipoproteins were used. The proteoliposomes were prepared by the cholate dialysis technique. In all proteoliposome preparations we found rouleaux structures and stacked discs. The particles formed with apolipoprotein A-I were the most homogeneous, followed by apolipoprotein D- and apolipoprotein C-I-containing particles. Apolipoprotein A-I was the most potent LCAT activator in our system followed by apolipoproteins C-I and D. The fractional esterification rate observed with apolipoprotein D-containing substrates amounted to 15-48% that of apolipoprotein A-I-containing ones. Neither apolipoprotein A-I- nor C-I-containing proteoliposomes gave linear reaction kinetics with LCAT. Even during the first 15-30 min of incubation, the kinetics deviated strikingly from linearity at all apolipoprotein concentrations. In contrast, proteoliposomes containing apolipoprotein D exhibited linear reaction kinetics up to 60-90 min. At low apolipoprotein A-I concentrations (5 micrograms/ml), the addition of apolipoprotein D to the incubates resulted in significantly higher esterification rates as compared to substrates containing apolipoprotein A-I only. This was not the case using substrates with high apolipoprotein A-I concentrations (50 micrograms/ml). From our results we speculate that apolipoprotein D may have some stabilizing effect on the enzyme LCAT.     

Effect of enzyme-matrix composition on potentiometric response to glucose using glucose oxidase immobilized on platinum

Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was immobilized in a crosslinked matrix of bovine serum albumin, catalase, glucose oxidase and glutaraldehyde on platinum foil. When placed in glucose solution, this enzyme-electrode elicited a potentiometric response that varied with the changes in glucose concentration. The immobilized glucose oxidase was present at 7.4-10.1 micrograms enzyme protein/ml of matrix, as determined with 125I-labelled enzyme. The coupled enzyme activity was stable over 120 h; however, the apparent activity of the immobilized glucose oxidase was markedly less than that for the same amount of enzyme free in solution. This indicated a significant level of diffusional resistance within the enzyme-matrix. The potentiometric response to glucose increased significantly as either the thickness of the enzyme-matrix or the glutaraldehyde content was reduced; this also was attributed to diffusional effects. Several enzyme-electrodes, constructed without exogenous catalase and with different amounts of glucose oxidase, showed greater sensitivity in potentiometric response at low glucose oxidase loadings. These results are consistent with the hypothesis that the potentiometric response arises from an interfacial reaction involving a hydrogen peroxide redox couple at a platinum surface. The data also suggest that an optimum range of hydrogen peroxide concentration exists for maximum electrode sensitivity.     

Reactions of Peroxynitrite and Nitrite with Organic Molecules and Hemoglobin

In this work, the reactions of nitrite (NO2-) and peroxynitrite (ONOO-) with organic molecules as well as with hemoglobin (Hb) were examined and the potential interference with the detection of hydrogen peroxide and Hb was investigated. ONOO- at low concentrations (35-140 microM) induced a concentration-dependent oxidation of o-phenylenediamine and guaiacol, and this process can be improved by the addition of Hb in a concentration-dependent manner. This enhancing effect of Hb was possibly due to the formation of such highly reactive species as ferrylHb during the reaction of ONOO- and Hb. NO2- also oxidized the aromatic amine o-phenylenediamine, but its efficiency was much lower than that of ONOO-. A 300-fold excess of NO2- over hydrogen peroxide inhibited the oxidation of Pyrogallol Red mediated by hydrogen peroxide and Hb, which was due in part to the reaction of NO2- with Hb ferryl species compound I and compound II and the phenoxyl radical. These data suggest that ONOO- and NO2- can interfere with the detection of hydrogen peroxide. The overestimation or underestimation of the hydrogen peroxide detected is dependent upon the organic molecule utilized for detection and the relative rate of NO2-, superoxide, and ONOO- generation.     

Ferrous ion-EDTA-stimulated phospholipid peroxidation. A reaction changing from alkoxyl-radical- to hydroxyl-radical-dependent initiation.

The stimulatory effect of ferrous salts on the peroxidation of phospholipids can be enhanced by EDTA when the concentration of Fe2+ in the reaction is greater than that of EDTA. Hydroxyl-radical scavengers do not inhibit peroxidation until the concentrations of Fe2+ and EDTA in the reaction are equal. Lipid peroxidation is then substantially initiated by hydroxyl radicals derived from a Fenton-type reaction requiring hydrogen peroxide. Superoxide radicals appear to play some role in the formation of initiating species.     

Examination of the reaction of fully reduced cytochrome oxidase with hydrogen peroxide by flow-flash spectroscopy

The reaction of cytochrome c oxidase with hydrogen peroxide has been of great value in generating and characterizing oxygenated species of the enzyme that are identical or similar to those formed during turnover of the enzyme with dioxygen. Most previous studies have utilized relatively low peroxide concentrations (millimolar range). In the current work, these studies have been extended to the examination of the kinetics of the single turnover of the fully reduced enzyme using much higher concentrations of peroxide to avoid limitations by the bimolecular reaction. The flow-flash method is used, in which laser photolysis of the CO adduct of the fully reduced enzyme initiates the reaction following rapid mixing of the enzyme with peroxide, and the reaction is monitored by observing the absorbance changes due to the heme components of the enzyme. The following reaction sequence is deduced from the data. (1) The initial product of the reaction appears to be heme a(3) oxoferryl (Fe(4+)=O(2)(-) + H(2)O). Since the conversion of ferrous to ferryl heme a(3) (Fe(2+) to Fe(4+)) is sufficient for this reaction, presumably Cu(B) remains reduced in the product, along with Cu(A) and heme a. (2) The second phase of the reaction is an internal rearrangement of electrons and protons in which the heme a(3) oxoferryl is reduced to ferric hydroxide (Fe(3+)OH(-)). In about 40% of the population, the electron comes from heme a, and in the remaining 60% of the population, Cu(B) is oxidized. This step has a time constant of about 65 micros. (3) The third apparent phase of the reaction includes two parallel reactions. The population of the enzyme with an electron in the binuclear center reacts with a second molecule of peroxide, forming compound F. The population of the enzyme with the two electrons on heme a and Cu(A) must first transfer an electron to the binuclear center, followed by reaction with a second molecule of peroxide, also yielding compound F. In each of these reaction pathways, the reaction time is 100-200 micros, i.e., much faster than the rate of reaction of peroxide with the fully oxidized enzyme. Thus, hydrogen peroxide is an efficient trap for a single electron in the binuclear center. (4) Compound F is then reduced by the final available electron, again from heme a, at the same rate as observed for the reduction of compound F formed during the reaction of the fully reduced oxidase with dioxygen. The product is the fully oxidized enzyme (heme a(3) Fe(3+)OH(-)), which reacts with a third molecule of hydrogen peroxide, forming compound P. The rate of this final reaction step saturates at high concentrations of peroxide (V(max) = 250 s(-)(1), K(m) = 350 mM). The data indicate a reaction mechanism for the steady-state peroxidase activity of the enzyme which, at pH 7.5, proceeds via the single-electron reduction of the binuclear center followed by reaction with peroxide to form compound F directly, without forming compound P. Peroxide is an efficient trap for the one-electron-reduced state of the binuclear center. The results also suggest that the reaction of hydrogen peroxide to the fully oxidized enzyme may be limited by the presence of hydroxide associated with the heme a(3) ferric species. The reaction of hydrogen peroxide with heme a(3) is very substantially accelerated by the availability of an electron on heme a, which is presumably transferred to the binuclear center concomitant with a proton that can convert the hydroxide to water, which is readily displaced.     

Steady-state kinetics of yeast cytochrome c peroxidase catalyzed oxidation of inorganic reductants by hydrogen peroxide

Rates of yeast cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) catalyzed oxidation of bis(tripyridine)cobalt(II) ion, penta(amine)pyridineruthenium(II) ion and ferrocyanide ion by hydrogen peroxide have been found to obey the empirical equation: (formula; see text) in the pH range 5 to 8, and at saturating H2O2 concentrations. [( S] and [CcP] are the concentrations of the reductant and the enzyme, respectively.) Values of k2 were found to be independent of the reductant. The term k0[S] is only significant with the cobalt and ruthenium complexes at high pH. The mechanism proposed to account for this rate equation differs significantly from previous mechanistic proposals. In particular, the rate data require the assignment of the rate-limiting step at high substrate concentrations to a slow electron-transfer within the enzyme, and not, as previously suggested, to saturation of substrate binding to the enzyme. Also, the term k0[S] implies that the reactive substrates, including the natural substrate (yeast cytochrome c), react with the hydrogen peroxide-heme complex and not with the radical species formed by reaction with hydrogen peroxide in the absence of reductants.     

On-line determination of glucose concentration throughout animal cell cultures based on chemiluminescent detection of hydrogen peroxide coupled with flow-injection analysis.

A flow-injection analysis (FIA) system for the on-line determination of glucose in animal cell cultures is described. The system is based on immobilized glucose oxidase (GOD). The hydrogen peroxide generated in the enzyme reaction is determined via a highly sensitive chemiluminescent reaction with luminol. Based on the measurement of the maximum emitted light intensity, the system was able to analyse hydrogen peroxide over the concentration range of 10(-7) to 10(-2) M. For glucose determination, the system has a linear range of 10(-5) to 5 x 10(-2) M glucose, with an r.s.d. of 3% at the 1 mM level (5 measurements). The influence of luminol and buffer concentrations, pH and temperature on the chemiluminescent reaction were investigated. The enzyme reactor used was stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per h. The system has been successfully applied to on-line monitoring of glucose concentration during an animal cell culture, designed for the production of human antithrombin III factor. Results obtained with the FIA system were compared with off-line results, obtained with a Yellow Springs Instrument Company Model 27 (YSI).