[Study on anemia and vitamin A and vitamin D nutritional status of Chinese urban pregnant women in 2010-2012].

Objective: To evaluate the prevalence of anemia and the nutritional status of vitamins A and D by analyzing hemoglobin, serum retinol, and serum 25-hydroxyvitamin D levels in Chinese urban pregnant women during 2010-2012. Methods: Data were obtained from the China Nutrition and Health Survey in 2010-2012. Using multi-stage stratified sampling and population proportional stratified random sampling, 2 250 pregnant women from 34 metropolis and 41 middle-sized and small cities were included in this study. Information was collected using a questionnaire survey. The blood hemoglobin concentration was determined using the cyanmethemoglobin method, and anemia was determined using the World Health Organization guidelines combined with the elevation correction standard. The serum retinol level was determined using high-performance liquid chromatography, and vitamin A deficiency (VAD) was judged by the related standard recommended by the World Health Organization. The vitamin D level was determined using enzyme-linked immunosorbent assay and vitamin D deficiency was judged by the recommendation standards from the Institute of Medicine of The National Academies. The hemoglobin, serum retinol, and serum 25-hydroxyvitamin D levels were compared, along with differences in the prevalence of anemia, VAD, and the vitamin D deficiency rate (including deficiency and serious deficiency). Results: A total of 1 738 cases of hemoglobin level, 594 cases of serum retinol level, and 1 027 cases of serum 25-hydroxyvitamin D were available for analysis in this study. The overall blood hemoglobin level (P(50) (P(25)-P(75))) was 122.70 (114.00-131.10) g/L; 123.70 (115.21-132.00) g/L for metropolis and 122.01 (113.30-130.40) g/L for middle-sized and small cities. The blood hemoglobin level of metropolis residents was significantly higher than that of middle-sized and small city residents (P=0.027). The overall prevalence of anemia was 17.0% (295/1 738). The overall serum retinol level (P(50) (P(25)-P(75))) was 1.61 (1.20-2.06) µmol/L; 1.50 (1.04-2.06) µmol/L for metropolis and 1.63 (1.31-2.05) µmol/L for middle-sized and small cities. The serum retinol level of metropolis residents was significantly higher than that of middle-sized and small city residents (P=0.033). The overall prevalence of VAD was 7.4% (47/639); 11.5% (33/286) for metropolis and 4.0% (14/353) for middle-sized and small cities. A significant difference was observed in the prevalence of VAD between metropolis and middle-sized and small city residents (P<0.001). The overall serum 25-hydroxyvitamin D level (P(50) (P(25)-P(75))) was 15.41 (11.79-20.23) ng/ml; 14.71 (11.15-19.07) ng/ml for metropolis and 16.02 (12.65-21.36) ng/ml for middle-sized and small cities. A significant difference was observed in the vitamin D level between metropolis and middle-sized and small city residents (P<0.001). The overall prevalence of vitamin D deficiency was 74.3% (763/1 027); A significant difference was observed in the prevalence of serious vitamin D deficiency between metropolis (30.64%(144/470)) and middle-sized and small city residents (26%(267/1 027))(P=0.002). There were no significant differences between blood hemoglobin level and the prevalence of anemia, VAD, and vitamin D deficiency. Conclusion: The prevalence of anemia in Chinese urban pregnant women improved from 2002 to 2012. The prevalence of vitamin D deficiency in pregnant women was generally more serious, while a certain percentage of women had VAD. The prevalence of VAD and serious vitamin D deficiency among pregnant women from metropolis was significantly higher than that of pregnant women from medium and small-sized cities.

Comparison of haemoglobin estimates using direct & indirect cyanmethaemoglobin methods.

BACKGROUND & OBJECTIVES: Estimation of haemoglobin is the most widely used method to assess anaemia. Although direct cyanmethaemoglobin method is the recommended method for estimation of haemoglobin, but it may not be feasible under field conditions. Hence, the present study was undertaken to compare indirect cyanmethaemoglobin method against the conventional direct method for haemoglobin estimation. METHODS: Haemoglobin levels were estimated for 888 adolescent girls aged 11-18 yr residing in an urban slum in Delhi by both direct and indirect cyanmethaemoglobin methods, and the results were compared. RESULTS: The mean haemoglobin levels for 888 whole blood samples estimated by direct and indirect cyanmethaemoglobin method were 116.1 ± 12.7 and 110.5 ± 12.5 g/l, respectively, with a mean difference of 5.67 g/l (95% confidence interval: 5.45 to 5.90, P<0.001); which is equivalent to 0.567 g%. The prevalence of anaemia was reported as 59.6 and 78.2 per cent by direct and indirect methods, respectively. Sensitivity and specificity of indirect cyanmethaemoglobin method were 99.2 and 56.4 per cent, respectively. Using regression analysis, prediction equation was developed for indirect haemoglobin values. INTERPRETATION & CONCLUSIONS: The present findings revealed that indirect cyanmethaemoglobin method overestimated the prevalence of anaemia as compared to the direct method. However, if a correction factor is applied, indirect method could be successfully used for estimating true haemoglobin level. More studies should be undertaken to establish agreement and correction factor between direct and indirect cyanmethaemoglobin methods.

[Study on the anemia status of Chinese urban residents in 2010-2012].

OBJECTIVE: The level of blood hemoglobin and the anemia status of Chinese urban residents in 2010-2012 was analyzed. METHODS: All the data in this study came from the China Nutrition and Health Survey in 2010-2012. By using multi-stage stratified sampling and population proportional stratified random sampling method, 74 276 residents aged above 6 from 34 metropolis and 41 middle-sized and small cities were included in this study. The concentration of blood hemoglobin was determined by cyanmethemoglobin method. Anemia was judged by the anemia standard recommended by WHO, combined with elevation correction standard. The level of blood hemoglobin, the prevalence of anemia and the 95%CI value were analyzed by using the complex sampling weighted processing, combined with the population figures released by the National Bureau of Statistics in 2009. RESULTS: In 2010-2012, the level of blood hemoglobin of Chinese city population was(144.16 ± 0.78)g/L, (152.88 ± 0.94)g/L for male and(135.01 ± 0.71)g/L for female, while (145.65 ± 1.22)g/L for metropolis and (143.90 ± 0.89)g/L for small and medium-sized. The anemia prevalence of Chinese city population (pregnant women were not included) was 9.7%(95%CI: 9.4%-10.1%), 6.8%(95%CI: 6.4%-7.3%) for male and 12.8%(95%CI: 12.2%-13.4%) for female, while 8.5%(95% CI: 8.0%-9.0%) for metropolis and 10.0%(95%CI: 9.5%-10.4%) for small and medium-sized. The anemia prevalence of 18-44 women (15.4%, 95%CI: 14.3%-16.6%) was the highest among all the age-groups, and the average anemia prevalence of people more than 60 years-old (including) (12.5%, 95%CI: 11.8%-13.2%) was higher than the other age-groups. CONCLUSION: The anemia prevalence of Chinese city population in 2010-2012 was obviously decreased in comparison of 10 years ago, while, more attention and improvement measures should be take upon women at reproductive age and the elder people.

Helix-Capping Histidines: Diversity of N-H···N Hydrogen Bond Strength Revealed by (2h)JNN Scalar Couplings.

In addition to its well-known roles as an electrophile and general acid, the side chain of histidine often serves as a hydrogen bond (H-bond) acceptor. These H-bonds provide a convenient pH-dependent switch for local structure and functional motifs. In hundreds of instances, a histidine caps the N-terminus of α- and 310-helices by forming a backbone NH···Nδ1 H-bond. To characterize the resilience and dynamics of the histidine cap, we measured the trans H-bond scalar coupling constant, (2h)JNN, in several forms of Group 1 truncated hemoglobins and cytochrome b5. The set of 19 measured (2h)JNN values were between 4.0 and 5.4 Hz, generally smaller than in nucleic acids (~6-10 Hz) and indicative of longer, weaker bonds in the studied proteins. A positive linear correlation between (2h)JNN and the difference in imidazole ring (15)N chemical shift (Δ(15)N = |δ(15)Nδ1 - δ(15)Nε2|) was found to be consistent with variable H-bond length and variable cap population related to the ionization of histidine in the capping and noncapping states. The relative ease of (2h)JNN detection suggests that this parameter can become part of the standard arsenal for describing histidines in helix caps and other key structural and catalytic elements involving NH···N H-bonds. The combined nucleic acid and protein data extend the utility of (2h)JNN as a sensitive marker of local structural, dynamic, and thermodynamic properties in biomolecules.

Nitric oxide scavenging by red blood cell microparticles and cell-free hemoglobin as a mechanism for the red cell storage lesion.

BACKGROUND: Intravascular red cell hemolysis impairs nitric oxide (NO)-redox homeostasis, producing endothelial dysfunction, platelet activation, and vasculopathy. Red blood cell storage under standard conditions results in reduced integrity of the erythrocyte membrane, with formation of exocytic microvesicles or microparticles and hemolysis, which we hypothesized could impair vascular function and contribute to the putative storage lesion of banked blood. METHODS AND RESULTS: We now find that storage of human red blood cells under standard blood banking conditions results in the accumulation of cell-free and microparticle-encapsulated hemoglobin, which, despite 39 days of storage, remains in the reduced ferrous oxyhemoglobin redox state and stoichiometrically reacts with and scavenges the vasodilator NO. Using stopped-flow spectroscopy and laser-triggered NO release from a caged NO compound, we found that both free hemoglobin and microparticles react with NO about 1000 times faster than with intact erythrocytes. In complementary in vivo studies, we show that hemoglobin, even at concentrations below 10 µmol/L (in heme), produces potent vasoconstriction when infused into the rat circulation, whereas controlled infusions of methemoglobin and cyanomethemoglobin, which do not consume NO, have substantially reduced vasoconstrictor effects. Infusion of the plasma from stored human red blood cell units into the rat circulation produces significant vasoconstriction related to the magnitude of storage-related hemolysis. CONCLUSIONS: The results of these studies suggest new mechanisms for endothelial injury and impaired vascular function associated with the most fundamental of storage lesions, hemolysis.

Plasma hemoglobin: A method comparison of six assays for hemoglobin and hemolysis index measurement.

INTRODUCTION: Plasma hemoglobin (Hb) is measured for assessment of in vivo and in vitro hemolysis. The objective of the present investigation was to conduct a method comparison of five quantitative and one semi-quantitative Hb and H-index (hemolysis index) assays to evaluate their performance measuring plasma Hb in clinical specimens. METHODS: One hundred and fourteen clinical specimens previously tested for plasma Hb using a laboratory-developed spectrophotometric assay were also tested for Hb using a HemoCue Plasma/Low Hb assay (azide methemoglobin), a laboratory-modified Pointe Scientific Hb assay (cyanmethemoglobin), tested for H-index measurements using a Roche cobas c501, an Abbott Architect c8000, and a semi-quantitative (binned) H-index measurement on a Beckman AU5800. The reference result was defined as the median Hb score (median of all Hb or H-index results). RESULTS: The laboratory-developed spectrophotometric Hb assay and Roche H-index methods mostly closely matched the median Hb score across all data, as well as for lower range median Hb score results ≤2.0 g/L. Two-way frequency table analysis using an Hb (or H-index) cutoff of 0.5 g/L (or 0.5 H-index units) was then performed to compare methods to the median Hb score cutoff. The Beckman method had the highest accuracy at this cutoff, the Roche and Abbott methods had the highest positive predictive value (PPV), and the Beckman, HemoCue, and Pointe methods had the highest negative predictive value (NPV). CONCLUSIONS: Plasma Hb and H-index results vary by method. Laboratories should evaluate the performance characteristics of their respective assays when considering adoption of spectrophotometric or chemical methods for plasma Hb assessment.

Hemoglobin redux: combining neutron and X-ray diffraction with mass spectrometry to analyse the quaternary state of oxidized hemoglobins.

Improvements in neutron diffraction instrumentation are affording the opportunity to re-examine the structures of vertebrate hemoglobins and to interrogate proton and solvent position changes between the different quaternary states of the protein. For hemoglobins of unknown primary sequence, structural studies of cyanomethemoglobin (CNmetHb) are being used to help to resolve sequence ambiguity in the mass spectra. These studies have also provided additional structural evidence for the involvement of oxidized hemoglobin in the process of erythrocyte senescence. X-ray crystal studies of Tibetan snow leopard CNmetHb have shown that this protein crystallizes in the B state, a structure with a more open dyad, which possibly has relevance to RBC band 3 protein binding and erythrocyte senescence. R-state equine CNmetHb crystal studies elaborate the solvent differences in the switch and hinge region compared with a human deoxyhemoglobin T-state neutron structure. Lastly, comparison of histidine protonation between the T and R state should enumerate the Bohr-effect protons.

Preliminary neutron and X-ray crystallographic studies of equine cyanomethemoglobin.

Room-temperature and 100 K X-ray and room-temperature neutron diffraction data have been measured from equine cyanomethemoglobin to 1.7 A resolution using a home source, to 1.6 A resolution on NE-CAT at the Advanced Photon Source and to 2.0 A resolution on the PCS at Los Alamos Neutron Science Center, respectively. The cyanomethemoglobin is in the R state and preliminary room-temperature electron and neutron scattering density maps clearly show the protonation states of potential Bohr groups. Interestingly, a water molecule that is in the vicinity of the heme group and coordinated to the distal histidine appears to be expelled from this site in the low-temperature structure.

Examination of blood haemoglobin concentration measured using the OSM2.

Blood haemoglobin concentration is regularly measured automatically by instruments reporting the value in around 1 min. The OSM2 from Radiometer is an example. Results from this instrument have been compared with those of a reference method using the hemiglobincyanide principle. Four healthy, moderately trained, young men (non-smokers) cycled for 2 min to exhaustion. Blood samples were drawn from indwelling catheters in the femoral artery and vein before exercise, during exercise and in the 1 h recovery. Blood haemoglobin concentration was analysed using both methods. The results of the OSM2 were linearly related to those of the control method, with a random variation of 0.14 mmol L(-1) (1.5%). For arterial blood, the OSM2 showed a systematic bias of -0.36 mmol L(-1) (-4%). For femoral venous samples the bias varied depending on the haemoglobin concentration, being negative at low concentrations and positive at high values (-3 to +2%). Consequently, the arteriovenous (a-v) difference differed systematically between the two methods. The varying bias in the results of the OSM2 for femoral-venous samples correlated with pH, pCO(2), O(2) saturation of haemoglobin (sO(2)) and with the haemoglobin concentration itself (cHb). Partial correlation analyses suggest that only the latter two correlations were independent, while correlations of the bias with pH and pCO(2) were removed when correcting for the effect of sO(2) and cHb. In conclusion, the OSM2 measures the blood haemoglobin concentration fairly precisely, but there is a variable bias of up to 4% in absolute value. Finally, the instrument does not report a-v differences reliably.

Zinc supplementation improved length growth only in anemic infants in a multi-country trial of iron and zinc supplementation in South-East Asia.

Data from 4 randomized, placebo-controlled, double-blind trials in Indonesia, Thailand, and Vietnam, the South-East Asian Multicountry Trial on Iron and Zinc supplementation in Infants (SEAMTIZI), were pooled to investigate the effects of iron and zinc supplementation infant growth. Infants (n = 2451) aged 4-6 mo old were supplemented with iron (10 mg/d) and/or zinc (10 mg/d) for 6 mo. Overall, neither iron nor zinc supplementation prevented the progressive growth faltering during infancy, which is common in many developing countries. However, infants who received zinc were less likely to be stunted at the end of the supplementation period (odds ratio 0.80; 95% CI 0.64-1.0). Boys had a 30% higher risk of being stunted at the end of the study than girls (P < 0.01). Baseline factors modified the effect of supplementation, with infants anemic at baseline (hemoglobin < 105 g/L) benefiting from zinc supplementation, with an estimated increase in height-for-age Z-score (HAZ) score of 0.17 (P < 0.01), but with no effect of zinc supplementation on growth in infants not anemic at baseline. Iron supplementation negatively affected linear growth in infants with a birth weight of >3500 g (estimated effect size, -0. 14 HAZ score; P < 0.01), but with no significant effect in infants with a lower birth weight. This study shows that blanket supplementation of infants with iron or zinc will not be beneficial to all recipients and may have adverse effects in some. Hence, interventions such as iron and zinc supplementation for infants should be restricted to subgroups in which there is a clear benefit and baseline factors should be considered and characterized before implementing new policies.

Association between magnesium : iron intake ratio and diabetes in Chinese adults in Jiangsu Province.

BACKGROUND: The intake of iron is positively related to the risk of diabetes, whilst magnesium intake is inversely related. However, it is unknown whether there is an interaction between dietary magnesium and iron expressed as a ratio and diabetes. METHODS: This is a cross-sectional household survey carried out in 2002 in Jiangsu Province, China. A total of 2849 men and women aged >or = 20 years participated (participation rate 89.0%). Iron and magnesium intake was assessed by 3-day weighed food records. Fasting plasma glucose, serum ferritin and haemoglobin were measured. RESULTS: The mean intake of iron and magnesium was 25 mg/day and 332 mg/day, respectively. The prevalence of diabetes was 3.0% in men and 2.6% in women. Magnesium intake was negatively associated with diabetes when adjusted for age and sex, but not in a fully adjusted model. A strong inverse association between magnesium : iron intake ratio and diabetes was observed. In the fully adjusted model, the odds ratios of diabetes across quartiles of magnesium : iron intake ratio were: 1.0, 0.63 [95% confidence interval 0.32-1.25], 0.36 (0.16-0.81) and 0.48 (0.20-1.14) (P for trend 0.038). There was an interaction between central obesity and magnesium : iron ratio. CONCLUSION: Magnesium : iron intake ratio is an independent risk marker for diabetes in Chinese adults. As this is a cross-sectional study, we cannot establish any causal relationship.

Comparative evaluation of haemoglobin estimation amongst pregnant women in Ibadan: Hemocue - B haemoglobin analyzer versus haemiglobincyanide (standard) method as the gold standard.

This was a comparative crossectional study that was conducted among 557 antenatal women at the University College Hospital, Ibadan, Nigeria between 1st April and 30th May 2006. The study compared the accuracy of haemoglobin estimation using an automated HemoCue B analyzer kit with a standard laboratory method (haemogloincyanide) as the gold standard. The result showed a positive correlation of HaemoCue haemoglobin results when compared with the standard methods (Pearson's correlation coefficient = 0.892; p value = 0.000). We concluded that the use of the automated HemoCue kit for haemoglobin estimation gives an objective assessment which overcomes the challenge of extra skilled manpower or power outages in rural areas thereby enhancing access to quality of care in resource poor countries.

Evaluation of microtitre plate-based Haemoglobin estimation.

INTRODUCTION: Haemoglobin estimation is one of the most important clinical investigations. Many techniques are available to measure haemoglobin; still there is a need for a haemoglobin assay technique which is cheap, robust and simple and can be used in field conditions very quickly using figure prick sample. We evaluated a cyanmethaemoglobin-based haemoglobin estimation using a microtitre plates for the purpose. METHODS: Microtitre plate-based haemoglobin estimation was developed using cyanmethaemoglobin-based assay and was compared with standard haematology analyser-based haemoglobin estimation in a large number of samples from a population of voluntary blood donors. Various tests were performed to evaluate the stability of colour, variation of the results during duplicate assay on the same days and on different days as well as linearity of the test was performed against broad range of haemoglobin values for the new microtitre plate-based technique. Standard statistical test of significance was applied to validate the assay. RESULTS: Total 200 samples from in-house and field conditions were evaluated. 10 µL blood sample in 300 µL Drabkin's solution provided optimum and comparable results after 10 minutes of incubation. The colour was stable up to 6 hours, the coefficient of variation was less than 3%, and the cost per test including everything was less than 3 cent/2P. Turnaround time for 90 samples was only 30 minutes. CONCLUSION: Cyanmethaemoglobin-based assay in microtitre plate is feasible, robust, rapid, cheap and cost-effective method for haemoglobin estimation in field conditions.

Determination of the formal redox potentials of the cyanhaemoglobin/cyanmethaemoglobin and the myoglobin/metmyoglobin couples at neutral pH.

Determination of a representative formal redox potential of the Fe(II)/Fe(III) redox couple in cyanhaemoglobin, at pH=7 and related to the state in solution, was the objective of this work. It was achieved at low concentrations of the protein (5µM) to circumvent undesired adsorption. Square-wave voltammetry instead of classical cyclic voltammetry was applied because this method is more sensitive and provides information on the formal redox potential and reversibility, even for rapid processes. We obtained E°'=-0.12±0.01V for cyanhaemoglobin and E°'=-0.10±0.01V, vs. SHE, for myoglobin in comparison. These values differ by only 20mV because the two Fe(II)/Fe(III) redox centres are embedded in closely resembling chemical environments. The small difference is probably owed to the additional axially coordinating cyanide ligand in cyanmethaemoglobin which slightly favours the Fe(III) state in the haem macrocycle.

Updated Method for In Vitro Analysis of Nanoparticle Hemolytic Properties.

Hemolysis is damage to red blood cells (RBCs), which results in the release of the iron-containing protein hemoglobin into plasma. An in vitro assay was developed and described earlier for the analysis of nanoparticle hemolytic properties. Herein, we present a revised version of the original protocol. In this protocol, analyte nanoparticles and controls are incubated in blood. Undamaged RBCs are removed by centrifugation and hemoglobin, released by the damaged erythrocytes, is converted to cyanmethemoglobin by incubation with Drabkin's reagent. The amount of cyanmethemoglobin in the supernatant is measured by spectrophotometry. This measured absorbance is compared to a standard curve to determine the concentration of hemoglobin in the supernatant. The measured hemoglobin concentration is then compared to the total hemoglobin concentration to obtain the percentage of nanoparticle-induced hemolysis. The revision includes updated details about nanoparticle sample preparation, selection of nanoparticle concentration for the in vitro study, updated details about assay controls and case studies about nanoparticle interference with the in vitro hemolysis assay.

Spontaneous oxygen radical generation by sickle erythrocytes.

Since the various membrane abnormalities of sickle erythrocytes might result from excessive accumulation of oxidant damage, we have measured the generation of superoxide, peroxide, and hydroxyl radical by normal and sickle erythrocytes using assays involving reduction of cytochrome c, aminotriazole inhibition of catalase, and methane evolution from dimethyl sulfoxide, respectively. Compared with normal erythrocytes, sickle erythrocytes spontaneously generate approximately twice as much superoxide, peroxide, and hydroxyl radical. One possible source of hydroxyl radical generation was identified as hemichrome, excessive amounts of which are bound to sickle erythrocyte membranes. Hemichrome did not generate hydroxyl radical when exposed to superoxide alone or peroxide alone. However, in the presence of both superoxide and peroxide, hemichrome greatly facilitated hydroxyl radical generation. Supporting this, normal erythrocyte membranes induced to acquire sickle hemichrome concomitantly acquired an enhanced ability to mediate hydroxyl radical generation. Finally, sickle erythrocyte membranes greatly enhanced superoxide/peroxide-driven hydroxyl radical generation as compared with normal erythrocyte membranes. These data suggest that an excessive accumulation of oxidant damage in sickle erythrocyte membranes might contribute to the accelerated membrane senescence of these cells. They further indicate that accumulation of oxidant damage could be a determinant of normal erythrocyte membrane senescence.

Evaluation of hemoglobin of blood donors deferred by the copper sulphate method for hemoglobin estimation.

INTRODUCTION: The copper sulphate (CuSO4) specific gravity test for Hb screening tends to give inappropriate failures. This prompted us to compare it with alternate screening methods. AIM: To study the impact of inaccuracy of CuSO4 method on donor deferral. METHODS: Capillary and venous blood samples of 400 potential blood donors failing the primary Hb screening using appropriately standardized CuSO4 test (specific gravity 1.053) were tested by Hemocue photometer, the Hb colour scale, Cyanmethemoglobin method as well as the automated hematology analyser, which was considered as the standard reference method. RESULTS: One hundred and sixteen donors (29%) who failed the CuSO4 test had true Hb levels >12.5 g/dl. The Hb levels of 131 (32.8%) deferred donors were between 12 and 12.5 g/dl. The sensitivity of Hemocue, Hb colour scale and Cyanmethemoglobin was 99%, 97% and 96% and their specificity was 45%, 93% and 46%, respectively. The positive predictive values (PPV) of Hemocue and Cyanmethemoglobin methods were low (43% and 44%, respectively) but their negative predictive values (NPV) were high (99%, and 97%, respectively). The Hb colour scale had an overall best performance with a PPV of 96% and NPV of 95%. CONCLUSION: The Hemoglobin colour scale which is inexpensive, convenient for field testing and has the overall best performance, is the most suitable for donor Hb screening. Since its readability is 12 g/dl, lowering the donor Hb threshold to 12 g/dl should be actively considered.

Visualizing the Bohr effect in hemoglobin: neutron structure of equine cyanomethemoglobin in the R state and comparison with human deoxyhemoglobin in the T state.

Neutron crystallography provides direct visual evidence of the atomic positions of deuterium-exchanged H atoms, enabling the accurate determination of the protonation/deuteration state of hydrated biomolecules. Comparison of two neutron structures of hemoglobins, human deoxyhemoglobin (T state) and equine cyanomethemoglobin (R state), offers a direct observation of histidine residues that are likely to contribute to the Bohr effect. Previous studies have shown that the T-state N-terminal and C-terminal salt bridges appear to have a partial instead of a primary overall contribution. Four conserved histidine residues [αHis72(EF1), αHis103(G10), αHis89(FG1), αHis112(G19) and ßHis97(FG4)] can become protonated/deuterated from the R to the T state, while two histidine residues [αHis20(B1) and ßHis117(G19)] can lose a proton/deuteron. αHis103(G10), located in the α1:ß1 dimer interface, appears to be a Bohr group that undergoes structural changes: in the R state it is singly protonated/deuterated and hydrogen-bonded through a water network to ßAsn108(G10) and in the T state it is doubly protonated/deuterated with the network uncoupled. The very long-term H/D exchange of the amide protons identifies regions that are accessible to exchange as well as regions that are impermeable to exchange. The liganded relaxed state (R state) has comparable levels of exchange (17.1% non-exchanged) compared with the deoxy tense state (T state; 11.8% non-exchanged). Interestingly, the regions of non-exchanged protons shift from the tetramer interfaces in the T-state interface (α1:ß2 and α2:ß1) to the cores of the individual monomers and to the dimer interfaces (α1:ß1 and α2:ß2) in the R state. The comparison of regions of stability in the two states allows a visualization of the conservation of fold energy necessary for ligand binding and release.

Resonance Raman studies of methemoglobin derivatives at room temperature and 77 K.

Raman spectra in preresonance with the Soret absorption band are reported for the following methemoglobin derivatives: cyanide, cyanate, thiocyanate, hydroxy-, azide, and fluoride methemoglobin at 285 K and 77 K. For the mixed-spin derivatives, Raman intensity is observed to shift from the high-spin marker band (approx. 1480 cm-1) to the low-spin marker band (approx. 1505 cm-1) upon cooling to 77 K. In addition, Raman spectra of cyanate methemoglobin were taken as a function of temperature, and the log of the intensity ratio I1480/I1505 was found to be a linear function of 1/T, indicating a thermally activated process. We interpret these results as observations of temperature-induced spin transitions. In the case of cyanate methemoglobin we find the enthalpy and entropy differences between the high-spin and low-spin states to be deltaH = 600 +/- 40 cal x mol-1 and deltaS = 4.7 +/- 0.7 cal x mol-1 x K-1. The high-spin to low-spin ratio for cyanate methemoglobin determined by our experiment disagrees with the value reported for magnetic susceptibility measurements.

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated, tetrameric beta-chain from human adult hemoglobin.

The solution molecular structure and the electronic and magnetic properties of the heme pocket of the cyanomet complex of the isolated beta-chain of human adult hemoglobin, HbA, have been investigated by homonuclear 2D (1)H NMR in order to assess the extent of assignments allowed by (1)H NMR of a homo-tetrameric 65-kDa protein, to guide the future assignments of the heterotetrameric complex of HbA, and to compare the structure of the beta-chain to the crystallographically characterized complexes that contains the beta-chain. The target residues are those that exhibit significant (>|0.2| ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 104 target residues ( approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than |0.2| ppm. These comprehensive assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the beta-chain in terms of the crystal coordinates of the beta-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated beta-chain relative to that in the intact R-state HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in the beta-subunit of HbCO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature 2D NMR spectra are crucial to effective assignments in the tetrameric cyanomet beta-chain and that this approach should be similarly effective in HbA.