Reactivities of hydroxylamine and sodium bisulphite with carbonyl-containing haems with the prosthetic groups of the erythrocyte green haemoproteins

DeFilippi, L J; Toler, Linda S.; Hultquist, Donald E.

doi:10.1042/bj1790151

Cited by 4 publications

(4 citation statements)

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“…(19,20). The green heme associated with this protein has been shown to be a highly conjugated and polar hemin, markedly different from protohemin (29,30). The green protein is easily distinguished spectrally from the protohemin-reductase complex.…”

Section: Resultsmentioning

confidence: 99%

“…Recently (18), we obtained evidence that NADPHdependent reductase from bovine erythrocytes is structurally and immunochemically identical to the protein component of a green heme-binding protein that also has been isolated from bovine erythrocytes (19). The green heme form of the protein is remarkable in that it undergoes rapid autoxidation and rapid reaction with peroxides (20).…”

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confidence: 99%

“…We also report that this protein is present in liver as well as in erythrocytes. Some of these data have been presented in abstract form (21,22 Bovine erythrocyte green heme-binding protein was purified as described (19). Antibodies to the green heme-binding protein were raised in young adult male New Zealand White rabbits as described (18).…”

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Characterization of NADPH-dependent methemoglobin reductase as a heme-binding protein present in erythrocytes and liver.

Xu¹,

Quandt²,

Hultquist³

1992

Proc. Natl. Acad. Sci. U.S.A.

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An NADPH-dependent reductase, first shown in the 1930s to catalyze the methylene blue-dependent reduction of methemoglobin in erythrocytes, has now been characterized as a high-affinity heme-binding protein and has been detected in liver. Highly purified bovine erythrocyte reductase binds protohemin to form a 1:1 complex with a Kd of 7 nM. Binding of protohemin completely inhibits reductase activity. Other tetrapyrroles and fatty acids also bind to the reductase and inhibit its activity. Protoporphyrin, hematoporphyrin, and coproporphyrin form 1:1 complexes withKd values ranging from 1 to 5 IzM. The inhibition constants for a number of saturated and unsaturated fatty acids range from 6 to 52 ,LM. A protein that is immunologically cross-reactive to the reductase has been detected in the cytosolic fractions of bovine and rat liver and of bovine, rat, rabbit, and human erythrocytes. By immunoblot analysis, the bovine liver and erythrocyte proteins appear identical in size, as do the rat liver and erythrocyte proteins. The concentration of the protein in bovine erythrocytes has been estimated by quantitative immunoblotting to be 10 juM. The detection of this protein in liver cells, the demonstration of its binding properties, and its weak reductase activity bring into question the long-held belief that this is uniquely an erythrocyte protein and that it functions as a reductase.Methylene blue stimulation of methemoglobin reduction in human erythrocytes was described 60 years ago by Warburg et al. (1), Steele and Spink (2), and Williams and Challis (3). Subsequent work by a number of investigators resulted in preparations of a cytosolic NADPH-dependent reductase that catalyzed the reduction of methylene blue and flavins and, in the presence of redox couplers, catalyzed the reduction of methemoglobin (4-9). Whereas this NADPHdependent reductase is believed to contribute very little to methemoglobin reduction under normal conditions (10), its catalysis of methemoglobin reduction in the presence of methylene blue or riboflavin is the basis for the use of these compounds as therapeutic agents in the treatment of congenital and toxic methemoglobinemia (11)(12)(13). The erythrocyte reductase has variously been referred to as NADPH dehydrogenase, diaphorase, methemoglobin reductase, and, most recently, flavin reductase.The reductase has been shown to bind small molecules. Isolation procedures generally yield two forms of the reductase, which exhibit very similar activities but different isoelectric points; one, but not the other, of these forms is reported to possess protein-bound NADP+ and a proteinbound yellow chromophore of unknown structure (7,8,(14)(15)(16). The isolation of the protein in the presence of flavin yields a flavoprotein (17). The protein isolated without the addition of flavin contains no flavin and yet exhibits full reductase activity.Recently (18) Bovine erythrocyte green heme-binding protein was purified as described (19). Antibodies to the green heme-binding protein were raised in young adult ...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of NADPH-dependent methemoglobin reductase as a heme-binding protein present in erythrocytes and liver.

Xu¹,

Quandt²,

Hultquist³

1992

Proc. Natl. Acad. Sci. U.S.A.

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“…FR is widely distributed in human tissues, but is most abundant in erythrocytes. Intriguingly, FR\BVR-B was first isolated as 'green haem binding protein', although the nature of the tetrapyrrole has not been completely characterized [17,18]. It has been shown that FR can provide free reduced flavins for the reduction of met-haemoglobin ; however, under normal conditions NADH : cytochrome b & reductase (EC 1.6.2.2) probably acts as the physiological reducing activity for met-haemoglobin [16].…”

Section: Introductionmentioning

confidence: 99%

Initial-rate kinetics of the flavin reductase reaction catalysed by human biliverdin-IXβ reductase (BVR-B)

Cunningham

Gore

Mantle

2000

Biochemical Journal

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The initial-rate kinetics of the flavin reductase reaction catalysed by biliverdin-IXbeta reductase at pH 7.5 are consistent with a rapid-equilibrium ordered mechanism, with the pyridine nucleotide binding first. NADPH binding to the free enzyme was characterized using stopped-flow fluorescence quenching, and a K(d) of 15.8 microM was calculated. Equilibrium fluorescence quenching experiments indicated a K(d) of 0.55 microM, suggesting that an enzyme-NADPH encounter complex (K(d) 15.8 microM) isomerizes to a more stable 'nucleotide-induced' conformation. The enzyme was shown to catalyse the reduction of FMN, FAD and riboflavin, with K(m) values of 52 microM, 125 microM and 53 microM, respectively. Lumichrome was shown to be a competitive inhibitor against FMN, with a K(i) of 76 microM, indicating that interactions with the isoalloxazine ring are probably sufficient for binding. During initial experiments it was observed that both the flavin reductase and biliverdin reductase activities of the enzyme exhibit a sharp optimum at pH 5 in citrate buffer. An initial-rate study indicated that the enzyme obeys a steady-state ordered mechanism in this buffer. The initial-rate kinetics in sodium acetate at pH 5 are consistent with a rapid-equilibrium ordered mechanism, indicating that citrate may directly affect the enzyme's behaviour at pH 5. Mesobiliverdin XIIIalpha, a synthetic biliverdin which binds to flavin reductase but does not act as a substrate for the enzyme, exhibits competitive kinetics with FMN (K(i) 0.59 microM) and mixed-inhibition kinetics with NADPH. This is consistent with a single pyridine nucleotide site and competition by FMN and biliverdin for a second site. Interestingly, flavin reductase/biliverdin-IXbeta reductase has also been shown to exhibit ferric reductase activity, with an apparent K(m) of 2.5 microM for the ferric iron. The ferric reductase reaction requires NAD(P)H and FMN. This activity is intriguing, as haem cleavage in the foetus produces non-alpha isomers of biliverdin and ferric iron, both of which are substrates for flavin reductase/biliverdin-IXbeta reductase.

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An isotopic method for determining chemically reactive lysine based on succinylation

Anderson

Quicke

1984

J Sci Food Agric

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A method is described for the routine succinylation of proteins using [i4C]succinic anhydride as a means of measuring free .+amino groups. Maximal succinylation was achieved using 6~ guanidine hydrochloride as protein solvent and an 80-fold molar excess of succinic anhydride relative to total lysine residues. Treatment with hydroxylamine (pH 13, 25"C, 5 min) removed unwanted 0-succinyl esters. Succinylated protein was precipitated with tnchloroacetic acid, residual label washed out with ethanol and the extent of labelling measured in a scintillation counter. The method gave close to theoretical values (n.s., P>0.05) for lysyl residues in egg white lysozyme, bovine haemoglobin, ovalbumin and bovine serum albumin but gave a low value with /3-lactoglobulin and an overestimate with insulin attributable to the relatively high contribution of a-relative to &-amino groups in this low molecular weight protein. The method gave good results for 12 soya protein samples and was shown to be very sensitive to 'isopeptide-type' heat damage in these samples. The results correlated well ( r = 0.91) with those obtained by the well established dye-bound lysine difference procedure whereas, as could be expected, both these methods correlated poorly with total lysine determinations ( r = 0.69 for succinic anhydride and r = 0.77 for the dye-binding method). The proposed method shows promise as a rapid procedure for the estimation of available lysine, but further studies are necessary to test its ability to measure nutritionally available lysine in all categories of heat damage.

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Reactivities of hydroxylamine and sodium bisulphite with carbonyl-containing haems with the prosthetic groups of the erythrocyte green haemoproteins

Cited by 4 publications

References 30 publications

Characterization of NADPH-dependent methemoglobin reductase as a heme-binding protein present in erythrocytes and liver.

Characterization of NADPH-dependent methemoglobin reductase as a heme-binding protein present in erythrocytes and liver.

Initial-rate kinetics of the flavin reductase reaction catalysed by human biliverdin-IXβ reductase (BVR-B)

An isotopic method for determining chemically reactive lysine based on succinylation

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