Minimal structural requirements for root effect: crystal structure of the cathodic hemoglobin isolated from the antarctic fish Trematomus newnesi.

The cathodic hemoglobin component of the Antarctic fish Trematomus newnesi (HbCTn) is a Root-effect protein. The interpretation of its functional properties in relation to its sequence is puzzling. Indeed, HbCTn sequence is characterized by an extremely low histidyl content, and in particular by the lack of His146beta and His69beta, which are believed to be important in Bohr and Root effects, respectively. Furthermore, previous analyses suggested that the local environment of Asp95alpha, Asp99beta, and Asp101beta should not be appropriate for the formation of Asp-Asp interactions, which are important for the Root effect. Here, we report the high-resolution crystal structure of the deoxy form of HbCTn. Our data provide a structural interpretation for the very low oxygen affinity of the protein and insights into the structural determinants of the Root effect protein. The structure demonstrates that the presence of Ile41alpha and Ser97alpha at the alpha1beta2 interface does not prevent the formation of the inter-Asp interactions in HbCTn, as previous studies had suggested. The present data indicate that the hydrogen bond formed between Asp95alpha and Asp101beta, which is stabilized by Asp99beta, is per se sufficient to generate the Root effect, and it is the minimal structural requirement needed for the design of Root-effect Hbs.

Sample pre-treatment for CO-oximetric determination of carboxyhaemoglobin in putrefied blood and cavity fluid.

Blood or blood-containing cavity fluid samples recovered from decomposed bodies in suspected carbon monoxide (CO) poisoning cases often have low total haemoglobin (tHb) levels or contain significant amounts of oily droplets, methaemoglobin (MetHb) and sulphaemoglobin (SHb), all conditions which render the validity of the measurements of carboxyhaemoglobin (HbCO) by commercially available CO-oximeters doubtful. Two sample pre-treatment methods, namely, centrifugal evaporation and addition of a commercially available standard haemoglobin solution containing a known level of HbCO, were developed to render such putrefied samples amenable to analysis by CO-oximeters. The methods developed were validated with samples of various degrees of putrefaction and various HbCO levels. The relative uncertainty of the determined HbCO levels as compared to their corresponding reference levels for the centrifugal evaporation method was approximately 5% and that for the standard addition method was <20%.

Cardiac damage in pediatric carbon monoxide poisoning.

BACKGROUND: Cardiovascular disorders including myocardial ischemia and heart failure have been described in both laboratory animals and humans following carbon monoxide poisoning. Carbon monoxide cardiotoxicity may be clinically occult and often remains undiagnosed because of the lack of overt symptoms and specific ischemic changes in the electrocardiogram. Routine myocardial necrosis markers have low diagnostic efficiency, particularly in patients with concomitant skeletal muscle necrosis or multiple organ failure complicating carbon monoxide poisoning. Carbon monoxide-induced cardiotoxicity has been investigated rarely in children. CASE REPORT: This paper describes carbon monoxide poisoning in a 12-year-old child who suffered from occult cardiac damage despite mild symptoms and low carboxy hemoglobin concentrations. Myocardial and mitral valve dysfunctions were observed, suggesting an ischemia-like syndrome. Cardiac damage was completely reversible within 1 month. CONCLUSION: This case report supports that a prolonged carbon monoxide exposure can cause cardiac damage in children even in the absence of specific symptoms, cerebral failure and high carboxyhemoglobin concentrations.

Interaction between carbon monoxide and ethanol in fire fatalities.

Impairment due to ethanol is clearly a risk factor in deaths due to fire. However, it is less clear whether there is a physiological interaction between ethanol and carbon monoxide (CO) that would alter the carboxyhemoglobin saturation level (COHb sat.) that accounts for death. In an attempt to explore this issue further, 196 fire fatalities investigated by the Office of the Chief Medical Examiner, State of Maryland over a 3-year period were examined. COHb sat. and blood ethanol concentrations (BAC) were tabulated. Twelve cases positive for therapeutic or abused drugs other than lidocaine or atropine were excluded; 184 cases were included. The data indicate that ethanol does not affect the COHb sat. that accounts for death, since the percentage of cases positive for ethanol at a given COHb range shows no trends. Therefore, we conclude that although ethanol remains a risk factor in fire fatalities, the risk appears to be related to the impairment that it produces as opposed to a direct interaction with CO.

Purification, crystallization and preliminary x-ray diffraction analysis of carboxyhaemoglobin-II from the fish Piaractus mesopotamicus (pacu).

Carboxyhaemoglobin-II isolated from the pacu (Piaractus mesopotamicus) has been crystallized and X-ray diffraction data were collected to 2.0 A resolution using synchrotron radiation. Crystals were characterized as belonging to the space group I23; preliminary structural analysis reveals the presence of one dimer in the asymmetric unit.

Is there an evolution in the epidemiology and follow-up of carbon monoxide poisoning victims?

A retrospective study was conducted to investigate for potential changes in the epidemiology of acute carbon monoxide (CO) poisoning and to evaluate the recommendations within the emergency department (ED) on therapy and neurological and social follow-up of these patients. One hundred and sixty-seven patients with a non-intentional CO poisoning in the time period from 1995 to 1997 were reviewed and compared with data of a similar patient population between 1988 and 1990. Both patient groups were matched for age, sex and comorbidity. Comparing the epidemiological data of the two groups, three main evolutions were observed: (1) there is a global reduction in ED admissions for CO poisoning; (2) the number of young victims has a tendency to decrease; (3) the most common CO source has become an insufficiently functioning stove instead of a defective gas water heater. Regarding treatment and follow-up strategies three conclusions could be drawn: (1) the recommendations for hyperbaric-oxygen therapy were strictly followed; (2) the neurological follow-up was poor and has to be ameliorated and revisited; (3) the systematic follow-up by the social worker on the other hand resulted in immediate actions to prevent a second CO poisoning. This recently elaborated protocol for social evaluation emphasizes the important function of the social worker in the ED and the importance of transmural follow-up organized by the ED.

Convenient method to purify hemoglobin.

A convenient method to purify Hb solution from outdated RBC has been established for the starting material of Hb-based blood substitutes. To prevent MetHb formation during the procedure, Hb in RBC was carbonylated in advance. Then RBC was mixed with organic solvent for hemolysis and centrifuged for removal of stroma. The resulting SFHb solution was heated at 60 degrees C and generated precipitates were removed out by centrifugation. The purity of Hb (25 g/dl) was confirmed by SDS-PAGE. IEF and oxygen binding property of the Hb solution also guaranteed its purity and no denaturation of Hb. This method is applicable to large scale production of the purified Hb for the starting material of Hb-based blood substitutes.

Purification of concentrated hemoglobin using organic solvent and heat treatment.

A simple method for obtaining a purified and concentrated hemoglobin (Hb) solution (25 g/100 ml) from human red blood cells has been established. To prevent MetHb formation during the purification procedure, Hb in red blood cells was carbonylated in advance, and then washed red blood cells were mixed with organic solvents such as diethyl ether or dichloromethane for hemolysis and removal of stroma. The Hb solution was isolated by centrifugation (1900g) with the high removal efficiency of phospholipid (> 99.8%). After the solution was heated (60 degrees C, 1 h), the precipitates were removed by centrifugation. The purity of Hb was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing and oxygen-binding properties of the obtained Hb solution demonstrated its purity and showed no denaturation of globin. This purification procedure is applicable to large-scale production of the purified Hb.

Speed of intersubunit communication in proteins.

To determine the speed of communication between protein subunits, time-resolved absorption spectra were measured following partial photodissociation of the carbon monoxide complex of hemoglobin. The experiments were carried out using linearly polarized, 10-ns laser pulses, with the polarization of the excitation pulse both parallel and perpendicular to the polarization of the probe pulse. The substantial contribution to the observed spectra from photoselection effects was eliminated by isotropically averaging the polarized spectra, allowing a detailed comparison of the kinetics as a function of the degree of photolysis. These results show that prior to 1 microsecond both geminate ligand rebinding and conformational relaxation are independent of the number of ligands dissociated from the hemoglobin tetramer, as expected for a two-state allosteric model. After this time the kinetics depend on the ligation state of the tetramer. The conformational relaxation at 10 microseconds can be interpreted in terms of the two-state allosteric model as arising from the R to T quaternary conformational change of both unliganded and singly liganded molecules. These results suggest that communication between subunits requires about 1 microsecond and that the mechanism of the communication which occurs after this time is via the R to T conformational change. The optical anisotropy provides a novel means of accurately determining the extinction coefficients of the transient photoproduct. The decay in the optical anisotropy, moreover, provides an accurate determination of the rotational correlation time of 36 +/- 3 ns.

Resolution of the lifetimes and correlation times of the intrinsic tryptophan fluorescence of human hemoglobin solutions using 2 GHz frequency-domain fluorometry.

We used 2 GHz harmonic content frequency-domain fluorescence to measure the intensity and the anisotropy decays from the intrinsic tryptophan fluorescence from human hemoglobin (Hb). The tryptophan intensity decays are dominated by a short-lived component which accounts for 35-60% of the total steady state intensity. The decay time of this short component varies from 9 to 27 ps and this component is sensitive to the ligation state of Hb. Our error analyses indicate the uncertainty is about +/- 3 ps. The intensity decays also show two longer lived components near 0.7 and 8 ns, which are probably due either to impurities or to Hb molecules in conformations which do not permit energy transfer. The anisotropy decays indicate the tryptophan residues in Hb are highly mobile, with apparent correlation times near 55 ps.

Purification of human hemoglobin valence intermediates by preparative immobilized pH gradients.

We compare three separation techniques for preparative purposes, i.e. ion-exchange chromatography on CM-cellulose, conventional isoelectric focusing in polyacrylamide gel slabs and immobilized pH gradients. The biological system used to test the three methods is a solution containing four hemoglobin (Hb) valence intermediates, i.e. metHb, oxyHb, (alpha + beta O2)2 and (alpha O2 beta +)2. The delta pI between the two valence intermediates is 0.04 pH units. Immobilized pH gradients give the best performance in terms of resolving power, total amount of protein which can be loaded and retention of biological activity by the protein (the latter assessed by determination of CO dissociation rates).

Demonstration of intermediates between deoxy and carbonmonoxy hemoglobins by anaerobic high-performance liquid chromatography.

Intermediary ferrous hemoglobins (Hb) partially liganded with carbon monoxide (CO) were separated using an anaerobic high-performance liquid chromatography system equipped with a cation-exchange column at room temperature. Separations were achieved by applying various mixtures of oxy- and CO-Hb A to the column. The mobile phase contained 3 mM sodium dithionite which deoxygenated the oxy hemes but left the CO hemes intact, thereby producing hemoglobins bound with various numbers of CO molecules. Spectrophotometric analyses of the eluent showed that hemoglobins were eluted in the order of deoxy hemoglobin, Hb(CO), Hb(CO)2 Hb(CO)3 and Hb(CO)4.

Determination of the dissociation constant of oligomeric proteins by size-exclusion high-performance liquid chromatography: application to human haemoglobin.

The measurement of protein retention volumes on a size-exclusion chromatographic column offers the possibility of determining dissociation constants for oligomeric proteins, as changes in the retention volume, depending on the concentration of the protein, are due to a dissociation equilibrium. The retention volume may be calibrated in terms of dissociation constant by using either extreme concentration conditions or chemical modifications that shift the equilibrium towards a single species. When zonal chromatography is used, the dilution during elution modifies the equilibrium state. In contrast, the saturation method permits the concentrations of the different species to be kept constant. These two methods were compared and the elution factor that must be used in zonal chromatography on high-performance size-exclusion columns (LiChrospher Diol) was obtained. The tetramer-dimer dissociation constants of normal and modified haemoglobins were measured by this method, and the results are in accordance with flash photolysis measurements.

Iron-carbon bond lengths in carbonmonoxy and cyanomet complexes of the monomeric hemoglobin III from Chironomus thummi thummi: a critical comparison between resonance Raman and x-ray diffraction studies.

Soret-excited resonance Raman spectroscopy yields direct information regarding the iron-carbon bonding interactions in the cyanomet and carbonmonoxy complexes of hemoglobin III from Chironomus thummi thummi (CTT III) in solution. By isotope exchange in cyanide (13CN-, C15N-, and 13C15N-) and carbon monoxide (13CO, C18O, and 13C18O), we have assigned the Fe(III)-CN- stretching at 453 cm-1, the Fe(III)-C-N- bending at 412 cm-1, the Fe(II)-CO stretching at 500 cm-1, the Fe(II)-C-O bending at 574 cm-1, and the C-O stretching at 1960 cm-1. The resonance Raman data, in conjunction with those obtained from heme model complexes with well-known Fe-C bond distances, strongly suggest that the Fe(III)-CN- bond (approximately 1.91 A) is longer (hence weaker) than the Fe(II)-CO bond (approximately 1.80 A). This result disagrees with those of x-ray crystallographic studies [Steigemann, W. & Weber, E. (1979) J. Mol. Biol. 127, 309-338] in which the Fe-C bond lengths were reported as 2.2 A in cyanomet and 2.4 A in carbonmonoxy CTT III. Based on Badger's rule and normal mode calculations, the x-ray data would lead to the prediction of 279 cm-1 for the Fe(II)-CO stretching frequency in CTT III . CO, which was not observed. On the other hand, we estimate the Fe-CO bond as approximately equal to 1.82 A, which is very similar to the 1.80-A value in human Hb . CO crystals. Furthermore, we have used isotope shift data to estimate the Fe-C-O angle as 169 +/- 5 degrees, somewhat larger than the 161 degrees value found by Steigemann and Weber. We therefore conclude that there must be errors in the x-ray crystallographic refinement for the ligand geometry in carbonmonoxy and cyanomet CTT III.

Reactivity of the amino groups of carbonmonoxyhemoglobin S with glyceraldehyde.

The inhibition of erythrocyte sickling in vitro by glyceraldehyde has been shown previously to result from a reduced gelation of deoxyhemoglobin S. Accordingly, the sites of Schiff base formation of the sugar aldehyde with hemoglobin have been determined by peptide of the protein after treatment of CO-saturated sickle cells with 10 mM [14C]glyceraldehyde for 90 min and reduction with NaBH4. About 23% of the glyceraldehyde incorporated into hemoglobin was present at Val-1(beta) with very little present at Val-1(alpha) (less than 5%. The distribution of [14C]glyceraldehyde between the hemoglobin chains was 60% in the beta chains and 40% in the alpha chains. The reactive lysine residues of the beta chain were Lys-82, Lys-59, and Lys-120 (45%, 20%, and 16% of the total 14C in the beta chains, respectively). The most reactive lysine residue of the alpha chain was Lys-16 where 75% of the total [14C]glyceraldehyde was present. The limited number of sites reactive with glyceraldehyde indicate some type of selectivity in the reaction of sugar aldehydes with hemoglobin.

Scanning gel chromatography: determination of transport parameters from dynamic profiles measured at low solute concentration.

Direct optical scanning of solute boundaries in large zone gel chromatography experiments provides an accurate means of determining boundary profile shapes and rates of motion. A method has been developed for correcting such boundaries to a constant time frame, eliminating the distortion which arises from finite column scanning rate. Centroids of the corrected profiles can be used to determine the partition cross section for the solute of interest. The partition cross section and flow rate determine translational motion within the column. The axial dispersion coefficient, L, which characterizes rate of boundary spreading may also be calculated from the profiles. In order to explore these procedures a study of four noninteracting solutes was conducted. Partition cross sections determined from rates of motion of boundary centroids were found to be in good agreement with those determined by the equilibrium saturation method on the same column. In order to explore the lowest concentration limits of the technique and to illustrate the boundary characteristics for a self-associating solute, a study of carboxyhemoglobin was conducted over a wide concentration range. From measurements at 220 nm the lowest concentration where useful data could be obtained was 2 micrograms per ml (0.12muM heme). These results establish validity of the procedures used in analyzing the rates of boundary transport and in studying solute transport over a wide range of conditions.