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PubMed1996

Infrared spectroscopy of the cyanide complex of iron (II) myoglobin and comparison with complexes of microperoxidase and hemoglobin.

The cyanide complex of FeIIMb prepared and maintained at temperatures below 0 degrees C is sufficiently stable to permit spectroscopic characterization and allow comparison with free HCN and other ferric and ferrous CN complexes. The visible absorption spectrum of FeIIMb-CN has a split alpha band maxima at 571 and 563 nm, suggesting distortion in the x-y plane of the porphyrin. FeIIMb-CN, like the CO complex, was found to be optically active by circular dichroism. The C-N stretching frequencies for the CN-ferrous complexes are very sensitive to parameters within the heme pocket. The values are as follows: FEIIMb at pH 8, 2057 cm-1 with a shoulder appearing at 2078 cm-1 at pH 5.6; FeIIMp, 2034 cm-1. In contrast, the frequencies for C-N stretch differ little among ferric heme complexes, ranging from 2123 to 2125 cm-1 for myoglobin, hemoglobin, and microperoxidase. These values compare with free HCN (2094 cm-1) or CN- (2080 cm-1). Quantum chemical modeling of the neutral iron-porphyrin complex with imidazole and cyanide and of its anion was used to explain the effects of the cyanide coordination and of iron reduction on the C-N stretching frequencies. The lower nu C-N for FeIIMb-CN relative to the ferric complex is attributed to the appearance of additional electron density on all the anti-bonding CN orbitals. The extra electron density was also used to explain that the band width of C-N stretching mode was greater in the ferrous complexes than in the ferric complex. Finally, the calculation shows that sigma donation weakens the Fe-C bond, in qualitative agreement with the spontaneous dissociation of CN- from FeIIMb at -5 degrees C. The sensitivity of CN complexes of ferrous heme proteins to the heme pocket environment and the ability to correlate spectroscopic parameters with calculated electron density suggest that infrared spectroscopy of the CN ligand is an appropriate tool to study ferrous heme proteins.

Animals↗

PubMed2001

Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on agonist-induced platelet aggregation and RANTES release in vitro.

We studied the effects of hemoglobin-vesicles modified with PEG (PEG-HbV), a type of liposome-encapsulated hemoglobin (LEH), on human platelet functions in vitro. The effect of a low concentration of PEG-HbV (Hb; 5.8 mg/dl) was assessed by examining an agonist-induced aggregation response, and that of relatively high concentrations of PEG-HbV (Hb; 0.29, 1 and 2 g/dl) by measuring the release of RANTES (Regulated upon activation, normal T-cell expressed and presumably secreted) from platelets, which is regarded as a marker of platelet activation. The preincubation of platelets with PEG-HbV at 5.8 mg/dl of Hb did not affect platelet aggregation induced by collagen, thrombin and ristocetin. The pretreatment of platelet-rich plasma (PRP) with PEG-HbV at concen trations up to 2 g/dl of Hb had no aberrant effects on the collagen-induced RANTES release. Furthermore, the collagen-induced release of RANTES from PRP was not affected by longer incubation with PEG-HbV at 2 g/dl of Hb. The basal levels of RANTES from PRP were unchanged in the presence of PEG-HbV. These results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on platelet functions in the presence of plasma.

Chemokine CCL5↗

PubMed1981

Iron deficiency in one-year-old infants: comparison of results of a therapeutic trial in infants with anemia or low-normal hemoglobin values.

The purpose of this study was to determine the Hgb response to a therapeutic trial of iron in infants with anemia compared to those with low-normal hemoglobin values. Hgb was determined in 1.128 one-year-old infants. The 278 infants (25%) who had an Hgb less than 11.5 gm/dl were given a three-month oral course of ferrous sulfate (3 mg iron/kg/day); the regimen was satisfactorily completed by 188 infants. Of the 75 infants whose initial capillary Hgb was in the anemic range (Hgb less than 11.0 gm/dl), 45% had an increase in venous Hgb greater than or equal to 1.0 gm/dl. Of the 113 infants with initial capillary values in the low-normal range (11.0 to 11.4 gm/dl), 28% had greater than or equal to 1.0 gm/dl Hgb response. Despite the lower rate of response in the low-normal group, almost half of the infants with a greater than or equal to 1 gm/dl response would have been missed by using the generally accepted cutoff value of 11 gm/dl for a therapeutic trial. Because of the low cost and simplicity of a therapeutic trial, we favor including the low-normal Hgb group for a therapeutic trial of iron in order to avoid missing iron-responsive individuals among groups of infants with a similarly high prevalence of iron deficiency anemia.

Adult↗

PubMed2002

Once weekly is superior to daily iron supplementation on height gain but not on hematological improvement among schoolchildren in Thailand.

Intermittent iron supplementation has been suggested as a replacement for daily iron supplements for reducing anemia in developing countries. The effects of once weekly and daily iron supplementation on hemoglobin (Hb), serum ferritin (SF), prevalence of anemia, weight and height are compared in this study. Primary schoolchildren (n = 397) from two selected schools in the Hat Yai rural area, southern Thailand, were recruited in 1999. All children received Albendazole and then randomly received ferrous sulfate (300 mg/tablet) either daily or weekly, or a placebo for 16 wk. The average increase in Hb was not significantly different between the daily (mean +/- SD; 6.5 +/- 6.0 g/L) and weekly (5.7 +/- 6.3 g/L) groups. However, the average increase in SF was greater (P < 0.01) in the daily (mean +/- SD; 39.8 +/- 30.3 microg/L) than the weekly (13.4 +/- 17.3 microg/L) group. All cases of iron deficiency anemia were abolished in both daily and weekly groups, whereas no reduction in prevalence occurred in the placebo group. Height gain was greater in children who received weekly (mean +/- SD; 2.6 +/- 0.9 cm) than in those who received daily iron (mean +/- SD; 2.3 +/- 0.8 cm), (P < 0.01). Weight gain, weight-for-age and height-for-age were not significantly different among the intervention groups. It is concluded that a weekly iron dose is more effective than a daily dose in height gain but not in hematological improvement over 16 wk of supplementation.

Adolescent↗

PubMed1995

Hemoglobin-induced lipid peroxidation in the retina: a possible mechanism for macular degeneration.

To investigate a possible link between subretinal hemorrhage and macular degeneration, oxyhemoglobin (HbO2) or methemoglobin (metHb) was incubated with retinal homogenate and unsaturated phospholipid peroxidation was monitored by (a) assay of thiobarbituric acid-reactive substances (TBARS), (b) luminescence originating from an energy transfer of lipid-degraded products to rose bengal, and (c) the decrease in composition of highly unsaturated fatty acids of phospholipids. TBARS formation and rose bengal luminescence in the case of metHb-induced lipid peroxidation were about 1.5 times greater than those in HbO2-induced lipid peroxidation. alpha-Tocopherol, a lipid-soluble antioxidant, and docosahexaenoic acid, a major unsaturated fatty acid, were slightly more rapidly decomposed after a 60-min incubation with metHb than with HbO2 at the same concentration. Atomic absorption analysis revealed that an equal concentration of iron was released from both HbO2 and metHb during incubation with retinal homogenates. The released iron may promote microsomal phospholipid peroxidation in the presence of endogenous ascorbate or NADPH-dependent cytochrome P-450 reductase because ascorbate oxidase and p-chloromercuribenzoic acid (an inhibitor of sulfhydryl enzymes) inhibited metHb- or HbO2-induced lipid peroxidation. MetHb-induced lipid peroxidation in retina was inhibited by KCN or NaN3, which binds to FeIII of metHb. KCN or NaN3 had no effect on HbO2-induced lipid peroxidation, because conversion of HbO2 to metHb, which can proceed in HbO2 incubated with phospholipid liposome, did not occur in retinal homogenates. It is concluded that metHb induces peroxidation of retinal unsaturated phospholipids (1) directly and (2) by releasing iron.

Animals↗

PubMed1988

Iron deficiency in female athletes: its prevalence and impact on performance.

The prevalence of iron deficiency and its impact on performance have not been adequately investigated in non-endurance athletes. This study evaluated these factors in 100 female intercollegiate athletes in various sports, and in 66 non-athletes. All subjects had determinations of hemoglobin, ferritin, and transferrin saturation. Athletes reported on diet, menses, and symptoms. Athletes and coaches described mood and performance. Iron-deficient athletes were treated with iron; others received placebo. The same data were collected at season's end. Initially 31/100 (31%) athletes had iron deficiency (ferritin less than 12 ng.ml-1; transferrin saturation less than 16%, or both) compared to 30/66 (45.5%) controls (not statistically significant). Compared to normal athletes, iron-deficient athletes did not have more symptoms of iron deficiency or differences in mood state, but they considered their performance to be worse (P less than 0.05). Their total iron intakes were similar, as were menstrual blood losses. At re-evaluation, 7/45 (15.6%) initially normal athletes were iron-deficient; 14/22 (63.6%) initially iron-deficient athletes were normal. Athletes receiving an iron supplement and their coaches did not report a greater improvement in performance or mood than athletes receiving a placebo. Female college athletes frequently had iron deficiency that could be successfully treated during the season, while some untreated normal athletes became iron-deficient. Iron deficiency and its treatment had no significant impact on symptoms or mood, but affected subjective assessment of performance.

Administration, Oral↗

PubMed1986

X-ray diffraction studies of a partially liganded hemoglobin, [alpha(FeII-CO)beta(MnII)]2.

We have applied single-crystal X-ray diffraction methods to analyze the structure of [alpha(FeII-CO)beta(MnII)]2, a mixed-metal hybrid hemoglobin that crystallizes in the deoxyhemoglobin quaternary structure (the T-state) even though it is half liganded. This study, carried out at a resolution of 3.0 A, shows that (1) the Mn(II)-substituted beta subunits are structurally isomorphous with normal deoxy beta subunits, and (2) CO binding to the alpha subunits induces small, localized changes in the T-state that lack the main directional component of the corresponding larger structural changes in subunit tertiary structure that accompany complete ligand binding to all four subunits and the deoxy to oxy quaternary structure change. Specifically, in the T-state, CO binding to the alpha heme group draws the iron atom toward the heme plane, and this in turn pulls the last turn of the F helix (residues 85 through 89) closer to the heme group. The direction of these small movements is almost perpendicular to the axis of the F helix. In contrast, when the structures of fully liganded and deoxyhemoglobin are compared, extensive structural changes occur throughout the F helix and FG corner, and the main component of the atomic movements in the F helix (in addition to the smaller component toward the heme) is in a direction parallel to the heme plane and toward the alpha 1 beta 2 interface. These findings are discussed in terms of the current stereochemical theories of co-operative ligand binding and the Bohr effect.

Cobalt↗

PubMed1983

Effects of dietary iron deficiency on muscle fiber characteristics and whole-body distribution of hemoglobin in mice.

Effects of dietary iron deficiency on growth, the distribution of hemoglobin (Hb) at rest and during exercise, the characteristics of muscle fiber types, and glycogen depletion patterns were studied in newly weaned male mice. Forty-eight mice were randomly divided into iron-deficient and control diet groups. Severe iron deficiency impaired general growth, but growth was restored following iron repletion. The mean +/- SEM blood Hb concentrations at rest after 7 weeks were 5.8 +/- 0.7 and 12.5 +/- 0.3 g/dl in iron-deficient and control groups, respectively. The mice fed iron-deficient diet for 7 weeks had an increased Hb level of 10.9 +/- 0.5 g/dl 1 week after an i.p. injection of Imferon (1.25 mg Fe). The Hb contents in brain and gastrocnemius as well as whole body were lowered by iron deficiency. Iron-deficient anemic mice tended to increase the percent distribution of Hb to brain during exercise. This value was significantly greater than in control and iron-treated groups. The iron-deficient group had relatively less glycogen than controls, but no significant tendency in glycogen depletion pattern was observed in any fiber types. It is suggested that decreased Hb content in working muscles due, in part, to greater distribution to brain could be one of the limiting factors for work performance in anemic individuals. It is further suggested that decrease in oxidative muscle fibers as well as the decreased concentration and/or activities of oxidative substances may also be one of the limiting factors.

Anemia, Hypochromic↗

PubMed2005

Role of CYP2E1 in the epoxidation of acrylamide to glycidamide and formation of DNA and hemoglobin adducts.

Acrylamide (AA) is an animal carcinogen, neurotoxin, and reproductive toxin. AA is formed in baked and fried carbohydrate-rich foods. Metabolism of AA occurs via epoxidation to glycidamide (GA) or direct conjugation with glutathione. Using CYP2E1-null mice, recent studies in this laboratory demonstrated that induction of somatic and germ cell mutagenicity in AA-treated mice is dependent on CYP2E1. We hypothesized that AA metabolism to GA is a prerequisite for the induction of AA-induced mutagenicity. Current studies were undertaken to assess the role of CYP2E1 in the epoxidation of AA to GA and the formation of DNA and hemoglobin (HGB) adducts. AA was administered to CYP2E1-null or wild-type mice at 50 mg/kg ip. Mice were euthanized 6 h later and blood and tissues were collected. Using LC-ES/MS/MS, AA, GA, and DNA- and HGB-adducts were measured. While the plasma levels of AA and GA were 115 +/- 14.0 and 1.7 +/- 0.31 microM in CYP2E1-null mice, they were 0.84 +/- 0.80 and 33.0 +/- 6.3 microM in the plasma of AA-treated wild-type mice. Administration of AA to wild-type mice caused a large increase in N7-GA-Gua and N3-GA-Ade adducts in the liver, lung, and testes. While traces of N7-GA-Gua adducts were measured in the tissues of AA-treated CYP2E1-null mice, these levels were 52- to 66-fold lower than in wild-type mice. Significant elevation of both AA- and GA-HGB adducts was detected in AA-treated wild-type mice. In AA-treated CYP2E1-null mice, levels of AA-HGB adducts were roughly twice as high as those in wild-type mice. In conclusion, current work demonstrated that CYP2E1 is the primary enzyme responsible for the epoxidation of AA to GA, which leads to the formation of GA-DNA and HGB adducts.

Acrylamide↗

PubMed2003

Comparison of the hemoglobin adducts formed by administration of N-methylolacrylamide and acrylamide to rats.

Acrylamide (AM) and N-methylolacrylamide (NMA) are used in the formulation of grouting materials. AM undergoes metabolism to a reactive epoxide, glycidamide (GA). Both AM and GA react with hemoglobin to form adducts that can be related to exposure to AM. The objective of this study was to evaluate the extent to which NMA could form the same adducts as AM. N-(2-carbamoylethyl)valine (AAVal derived from AM) and N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal derived from GA) were measured following a single oral dose of AM (50 mg/kg) or NMA (71 mg/kg) in male F344 rats. AAVal and GAVal were measured by a modified Edman degradation to produce phenylthiohydantoin derivatives and liquid chromatography/tandem mass spectrometry. In AM-treated rats, AAVal was 21 +/- 1.7-pmol/mg globin (mean +/- SD, n = 4), and GAVal was 7.9 +/- 0.8 pmol/mg. In NMA-treated rats, AAVal was 41 +/- 4.9 pmol/mg, and GAVal was 1.4 +/- 0.1 pmol/mg. Whether AAVal was derived from reaction of NMA with globin followed by loss of the hydroxymethyl group, or loss of the hydroxymethyl group to form AM prior to reaction with globin, is not known. However, the higher ratio of AAVal:GAVal in NMA-treated rats (29 vs. 2.6 in AM-treated rats) suggests that reaction of NMA with globin is the predominant route to AAVal in NMA-treated rats. The detection of GAVal in NMA-treated rats indicates oxidation of NMA, either directly or following conversion to AM. The lower levels of GAVal on NMA administration suggest that a much lower level of epoxide was formed than compared with AM treatment.

Acrylamide↗

PubMed1978

Magnetic circular dichroism studies of myoglobin, hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives.

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q00- and QV-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q00-band to the A-term in the QV-band. The evidneces are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the "hemoglobin-like" MCD, while the alkaline ferroperoxidase is characterized by the "myoglobin-like" MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoporteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at approximately 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at approximately 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-iron charge-transfer electronic transition which may be assigned as b( pi) leads to 3d. This charge-transfer band is strongly overlapped with usual B(pi --pi*) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.

Circular Dichroism↗

PubMed1985

Iron deficiency among incarcerated juvenile delinquents.

A population of 163 incarcerated delinquents (126 males and 37 females aged 12-18 years) was studied to determine the prevalence of iron deficiency and to compare hemoglobin (Hgb), mean corpuscular volume (MCV), serum ferritin (SF), and erythrocyte protoporphyrin/hemoglobin (EP/Hgb) as predictors of response to iron therapy. Thirty-two percent of females and 6% of males had SF less than or equal to 12 ng/ml; 51% of females and 24% of males had SF less than or equal to 20 ng/ml. The mean SF was 17.7 ng/ml for females and 29.2 ng/ml for males. Of the 163 subjects, 53 were at risk for iron deficiency based on SF, Hgb, EP/Hgb, or MCV criteria. Twenty-one completed treatment with iron, and nine had greater than 1 g rise in Hgb. The following tests identified responders: SF less than or equal to 12 ng/ml-5/9; SF less than or equal to 20 ng/ml-9/9; Hgb less than or equal to third percentile-4/9; Hgb less than or equal to tenth percentile-7/9; MCV less than or equal to tenth percentile-2/9; EP/Hgb greater than or equal to 3.0 micrograms/g Hgb-2/9; EP/Hgb greater than or equal to 2.5 micrograms/g Hgb-4/9. Serum ferritin less than or equal to 20 ng/ml had a false positive rate of 57%; Hgb less than or equal to tenth percentile and EP/Hgb greater than or equal to 2.5 mu g/g Hgb had no false positives. The significance of the high prevalence of iron deficiency among a population of incarcerated adolescents is not clear.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1997

Serum erythropoietin during normal pregnancy: relationship to hemoglobin and iron status markers and impact of iron supplementation in a longitudinal, placebo-controlled study on 118 women.

Serum erythropoietin (EPO) and its relationship to hemoglobin (Hb), iron status markers and iron supplementation during normal pregnancy was assessed in a longitudinal, placebo-controlled study on 118 women, 61 took daily tablets containing 66 mg ferrous iron from the second trimester until delivery and 57 took placebo. Blood samples were obtained at 4-week intervals until delivery as well as post-partum. In the placebo-treated women, median serum EPO rose from 22.5 U/l at inclusion to 35.0 U/l at delivery (P = 0.0001). In the iron-treated women, median serum EPO rose from 23.9 to 29.9 U/l (P = 0.0001). Serum EPO showed a steeper increase in the placebo-treated women than in the iron-treated women (P < 0.05). After delivery, serum EPO became normal in both groups (P = 0.0001). Median Hb was lower in placebo-treated (iron depleted) than in iron-treated (iron repleted) women (P < 0.05). In the placebo-treated women there was a negative correlation and in the iron-treated women a positive correlation between serum EPO and Hb. In the placebo-treated women, inverse correlations existed between serum EPO and serum transferrin saturation and serum ferritin, reflecting the consequences of iron deficiency, whereas the iron-treated women displayed no correlation. A physiological, nonhypoxia-induced increase in EPO production accounts for the basic expansion of the red cell mass during pregnancy. In placebo-treated women, iron deficient erythropoiesis constitutes an additional hypoxic stimulus, which induces a further increase in serum EPO.

Anemia, Hypochromic↗

PubMed1981

Diagnosis of iron deficiency: the limitations of laboratory tests in predicting response to iron treatment in 1-year-old infants.

This study was designed to compare the effectiveness of laboratory tests for iron deficiency (mean corpuscular volume, erythrocyte protoporphyrin, transferrin saturation, and serum ferritin) in predicting hemoglobin response to iron therapy in infants found to have low Hgb concentrations. Screening for anemia was performed on capillary blood of 1,128 healthy 1-year-old infants of United States Air Force personnel. The 25% who had Hgb values less than 11.5 gm/dl were asked to return for tests on venous blood before therapy and again after three months of therapy. Of the 188 infants completing therapy, 66 (35%) had a rise in Hgb concentration greater than or equal to 1.0 gm/dl and were designated responders. None of the confirmatory tests on venous blood reliably distinguished responders from those who subsequently showed no response. By using any one of the tests in combination with a capillary Hgb value less than 11.5 gm/dl, more than half of the infants with an abnormal value responded. But well over half of the responders would have been missed if treatment had been restricted to infants with abnormal values. Neither changes in the criteria for normality nor combinations of tests substantially improved our ability to distinguish the two groups. Because of the difficulty in distinguishing responders from nonresponders with additional laboratory tests and because of the simplicity, low cost, and relative safety of iron therapy in infants, we favor an initial therapeutic trial of iron first for determining the cause of low Hgb values in similar high-risk populations. Further costly workup can then be reserved for the small number of infants who still have unexplained Hgb concentrations less than 11.0 gm/dl after a therapeutic trial.

Anemia, Hypochromic↗

PubMed2004

NO binding induced conformational changes in a truncated hemoglobin from Mycobacterium tuberculosis.

The resonance Raman spectra of the NO-bound ferric derivatives of wild-type HbN and the B10 Tyr --> Phe mutant of HbN, a hemoglobin from Mycobacterium tuberculosis, were examined with both Soret and UV excitation. The Fe-N-O stretching and bending modes of the NO derivative of the wild-type protein were tentatively assigned at 591 and 579 cm(-1), respectively. Upon B10 mutation, the Fe-NO stretching mode was slightly enhanced and the bending mode diminished in amplitude. In addition, the N-O stretching mode shifted from 1914 to 1908 cm(-1). These data suggest that the B10 Tyr forms an H-bond(s) with the heme-bound NO and causes it to bend in the wild-type protein. To further investigate the interaction between the B10 Tyr and the heme-bound NO, we examined the UV Raman spectrum of the B10 Tyr by subtracting the B10 mutant spectrum from the wild-type spectrum. It was found that, upon NO binding to the ferric protein, the Y(8a) mode of the B10 Tyr shifted from 1616 to 1622 cm(-1), confirming a direct interaction between the B10 Tyr and the heme-bound NO. Furthermore, the Y(8a) mode of the other two Tyr residues at positions 16 and 72 that are remote from the heme was also affected by NO binding, suggesting that NO binding to the distal site of the heme triggers a large-scale conformational change that propagates through the pre-F helix loop to the E and B helices. This large-scale conformational change triggered by NO binding may play an important role in regulating the ligand binding properties and/or the chemical reactivity of HbN.

Bacterial Proteins↗

PubMed2001

Monomer-dimer equilibrium and oxygen binding properties of ferrous Vitreoscilla hemoglobin.

The monomer-dimer equilibrium and the oxygen binding properties of ferrous recombinant Vitreoscilla hemoglobin (Vitreoscilla Hb) have been investigated. Sedimentation equilibrium data indicate that the ferrous deoxygenated and carbonylated derivatives display low values of equilibrium dimerization constants, 6 x 10(2) and 1 x 10(2) M(-1), respectively, at pH 7.0 and 10 degrees C. The behavior of the oxygenated species, as measured in sedimentation velocity experiments, is superimposable to that of the carbonylated derivative. The kinetics of O(2) combination, measured by laser photolysis at pH 7.0 and 20 degrees C, is characterized by a second-order rate constant of 2 x 10(8) M(-1) s(-1) whereas the kinetics of O(2) release at pH 7.0 is biphasic between 10 and 40 degrees C, becoming essentially monophasic below 10 degrees C. Values of the first-order rate constants (at 20 degrees C) and of the activation energies for the fast and slow phases of the Vitreoscilla Hb deoxygenation process are 4.2 s(-1) and 19.2 kcal mol(-1) and 0.15 s(-1) and 24.8 kcal mol(-1), respectively. Thus the biphasic kinetics of Vitreoscilla Hb deoxygenation is unrelated to the association state of the protein. The observed biphasic oxygen release may be accounted for by the presence of two different conformers in thermal equilibrium within the monomer. The two conformers may be assigned to a structure in which the heme-iron-bound ligand is stabilized by direct hydrogen bonding to TyrB10 and a structure in which such interaction is absent. The slow interconversion between the two conformers may reflect a very large conformational rearrangement in the disordered distal pocket segment connecting helices C and E.

Bacterial Proteins↗

PubMed2000

Ligand binding in the ferric and ferrous states of Paramecium hemoglobin.

The unicellular protozoan Paramecium caudatum contains a monomeric hemoglobin (Hb) that has only 116 amino acid residues. This Hb shares the simultaneous presence of a distal E7 glutamine and a B10 tyrosine with several invertebrate Hbs. In the study presented here, we have used ligand binding kinetics and resonance Raman spectroscopy to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands. In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition takes place with a pK(a) of approximately 9.0. The oxygen affinity (P(50) = 0.45 Torr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hydrogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy complex is an equilibrium mixture of a hydrogen-bonded closed structure and an open structure. Oxygen will dissociate preferentially from the open structure, and therefore, the fraction of open structure population controls the rate of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the higher on- and off-rates for CO relative to those in myoglobin. A high rate of ligand binding is also consistent with the observation of an Fe-histidine stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain. We postulate that the function of Paramecium Hb is to supply oxygen for cellular oxidative processes.

Amino Acid Sequence↗

PubMed1997

Comparison of styrene-7,8-oxide adducts formed via reaction with cysteine, N-terminal valine and carboxylic acid residues in human, mouse and rat hemoglobin.

The reactive metabolite of styrene, styrene-7,8-oxide (SO), reacts with a variety of nucleophilic sites in hemoglobin (Hb) to form SO-Hb adducts. Following the in vitro incubation of SO with blood from humans, NMRI mice and Sprague-Dawley rats, the second-order reaction rate constants were determined for the reaction of SO with cysteine (through both the alpha- and beta-carbons of SO), N-terminal valine (through the beta-carbon of SO), and carboxylic acid (presumably through both the alpha- and beta-carbons of SO) residues in Hb. The rate constants for cysteine adducts vary dramatically between species [2.04, 10.7, 133 L (mol Hb)-1 h-1 (alpha binding) for humans, mice and rats, respectively] and [0.078, 2.16, 20.4 L (mol Hb)-1 h-1 (beta binding), respectively]. The considerably higher rate of reaction with cysteine in rat Hb probably reflects the presence of an additional cysteine residue at position beta 125. Although the rate constants for valine adducts (1.82, 0.80, 0.29 L (mol Hb)-1 h-1, respectively) and COOH adducts (3.55, 1.94, 2.37 L (mol Hb)-1 h-1, respectively) are much more consistent, the inter-species differences are statistically significant for the reaction of SO with the N-terminal valine of Hb. Following the i.p. administration of styrene to mice and styrene and SO to rats, the levels of adducts at each of these sites were used in conjunction with the calculated rate constants to predict the integrated blood doses of SO. While the SO doses predicted from cysteine and valine adducts were very similar, that based upon COOH-binding was significantly different, presumably due to the instability of SO-COOH adducts. This research affirms the use of both cysteine and valine adducts, but not carboxylic acid adducts, as biomarkers of exposure to styrene and SO.

Animals↗