Hemoglobin Beta Chain Research Articles

Identification of a S-hexahydro-1H-azepine-1-carbonyl adduct produced by molinate on rat hemoglobin beta(2) and beta(3) chains in vivo.

Molinate is a thiocarbamate herbicide used in the rice industry for over 25 years, and regulatory reports have shown that administration of molinate results in reproductive toxicity in male rats. Previous in vitro studies indicate that molinate undergoes oxidative metabolism, forming reactive electrophilic intermediates capable of undergoing nucleophilic addition by protein nucleophiles. On the basis of in vitro studies, carbamylation of an active site serine residue in Hydrolase A has been proposed to be the mechanism responsible for the observed testicular toxicity. The experiments presented here utilize hemoglobin to characterize covalent protein modifications produced in vivo by molinate. Rats were dosed intraperitoneally with molinate as a function of exposure duration. Examination of globin from molinate-treated rats by HPLC demonstrated a new peak in the isolated samples and, when collected and analyzed using MALDI-TOF MS, revealed a 126 Da increase in mass relative to the native beta(3) chain. Digestion of the globin using Glu-C and analysis by MALDI-TOF MS revealed two modified peptide fragments at m/z 2743 and 4985 consistent with a 126 Da increase to peptide fragments [122-146] and [102-146] in the unmodified beta(2) and beta(3) chains of globin. Using selected reaction monitoring LC/MS/MS, S-hexahydro-1H-azepine-1-carbonyl cysteine (HHAC-Cys) was identified in the globin hydrolysates isolated from the molinate-treated rats, but not in the control samples, and the quantity of adduct exhibited a cumulative dose response. These experiments demonstrate the ability of molinate to covalently modify proteins in vivo in a dose dependent manner. For hemoglobin this modification was a carbamylation at Cys-125 similar to the modification produced by disulfiram and N,N-diethyldithiocarbamate. The ability of molinate to covalently modify cysteine residues provides a potential mechanism to account for enzyme inhibition following molinate exposure and suggests that enzymes with cysteine residues in their active site may be inhibited by molinate.

The hemoglobin system of the brown moray Gymnothorax unicolor: structure/function relationships.

The Gymnothorax unicolor hemoglobin system is characterized by two components, called cathodic and anodic on the basis of their isoelectric point, which were separated by ion-exchange chromatography. The oxygen-binding properties of the purified components were studied in the absence and presence of chloride and/or GTP or ATP in the pH range 6.5-8.0. Stripped cathodic hemoglobin showed a small reverse Bohr effect, high oxygen affinity, and low co-operativity; the addition of chloride only caused a small decrease in oxygen affinity. In the presence of GTP or ATP, the oxygen affinity was dramatically reduced, the co-operativity increased, and the reverse Bohr effect abolished. Stripped anodic hemoglobin is characterized by both low oxygen affinity and co-operativity, and displayed a normal Bohr effect; the addition of chloride increased co-operativity, whereas ATP and GTP significantly modulated oxygen affinity at acidic pH values, enhancing the Bohr effect and giving rise to the Root effect. The complete amino-acid sequences of the alpha and beta chains of both hemoglobins were established; the molecular basis of the functional properties of the hemoglobins is discussed in the light of the primary structure and compared with those of other fish hemoglobins.

Novel therapies for prevention of erythrocyte dehydration in sickle cell anemia.

Sickle cell anemia is a genetic disorder characterized by mutant hemoglobin (Hb) polymerization and resultant cell deformation (sickling) under conditions of reduced oxygen tension. The disease is caused by mutation of wild-type Glu to Val in position 6 of the beta-chain of hemoglobin, yielding hemoglobin S (HbS). The sickling process is markedly accelerated when the intracellular concentration of HbS is increased. A variable fraction of dehydrated erythrocytes is seen in the majority of patients, and these cells are believed to play an important role in the pathophysiology of the vasoocclusive events of sickle cell disease. Therapy of sickle cell disease is extremely limited in range and efficacy. Many patients still receive treatment only for symptomatic relief of sickle crises, painful episodes due to vasoocclusion by sickled cells. The last 15 years, however, have seen the identification of the principal transport pathways that mediate sickle erythrocyte dehydration, and the last 6 years have witnessed promising clinical tests of specific inhibitors of these pathways, with the intent of reducing cell sickling via inhibition of red cell dehydration. This review discusses the pathophysiology of sickle cell dehydration and explores current and future treatment options for in vivo prevention of sickle cell dehydration.

Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization--mass spectrometry, nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry, and tandem mass spectrometry.

Optimal application of biological mass spectrometry (MS) in combination with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of human cerebrospinal fluid (CSF) can lead to the identification of new potential biological markers of neurological disorders. To this end, we analyzed a number of 2-D PAGE protein spots in a human CSF pool using spot co-localization, N-terminal sequencing, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry (nanoLC-ESI-TOF-MS) with tandem MS switching. Our constructed CSF master contained 469 spots after image analysis and processing of 2-D gels. Upon visual inspection of our CSF master with the CSF pattern available on the ExPASy server, it was possible to locate and annotate 15 proteins. N-terminal sequence analysis and MALDI-MS peptide mass fingerprint analysis of both silver- and Coomassie Brilliant Blue (CBB) G-250-stained protein spots after in situ trypsin digest not only confirmed nine of the visually annotated spots but additionally resolved the identity of another 13 spots. Six of these proteins were not annotated on the 2-D ExPASy map: complement C3 alpha-chain (1321-1663), complement factor B, cystatin C, calgranulin A, hemoglobin beta-chain, and beta-2-microglobulin. It was clear that MALDI-MS identification from CBB G-250-stained, rather than from silver-stained, spots was more successful. In cases where no N-terminal sequence and/or no clear MALDI-MS result was available, nanoLC-ESI-TOF-MS and tandem MS automated switching was used to clarify and/or identify these protein spots by generating amino acid sequence tags. In addition, enrichment of the concentration of low-abundant proteins on 2-D PAGE was obtained by removal of albumin and immunoglobulins from the CSF pool using affinity chromatography. Subsequent analysis by 2-D PAGE of the fractionated CSF pool showed various new silver-stainable protein spots, of which four were identified by nanoLC-ESI-TOF-MS and tandem MS switching. No significant homology was found in either protein or DNA databases, indicating than these spots were unknown proteins.

Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin α and β chains

The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, conserved in mammalian and avian hemoglobins, is located near the functionally important alpha1-beta2 interface and C-terminal region of the beta chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4, 4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala-->Cys at 88alpha (rHb A88alphaC), Cys-->Ala at 93beta (rHb C93betaA) and Cys-->Thr at 93beta (rHb C93betaT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88alphaC compared with 2.2 for Hb A, whereas those for rHb C93betaA and rHb C93betaT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88alphaC was smaller than that in Hb A. Our experimental data have shown that the residues at 88alpha and 93beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed.

1F1345 ヘモグロビンβ鎖におけるLeu28の酸素親和性制御メカニズムの解明

1F1345 ヘモグロビンβ鎖におけるLeu28の酸素親和性制御メカニズムの解明

Identification and Characterization of Falcilysin, a Metallopeptidase Involved in Hemoglobin Catabolism within the Malaria Parasite Plasmodium falciparum

The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in the hemoglobin proteolytic pathway. It is unable to cleave hemoglobin or denatured globin but readily destroys peptide fragments of hemoglobin. Falcilysin cleavage sites along the alpha and beta chains of hemoglobin are polar in character, with charged residues located in the P1 and/or P4' positions. In contrast, plasmepsins I and II and falcipain prefer hydrophobic residues around the scissile bond. The gene encoding falcilysin has been cloned. Its coding sequence exhibits features characteristic of clan ME family M16 metallopeptidases, including an "inverted" HXXEH active site motif. Falcilysin shares primary structural features with M16 family members such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin as well as with data base hypothetical proteins that are potential M16 family members. The characterization of falcilysin increases our understanding of hemoglobin catabolism in P. falciparum and the unusual M16 family of metallopeptidases.

A biochemical and biophysical characterization of recombinant mutants of fetal hemoglobin and their interaction with sickle cell hemoglobin.

Three recombinant mutants of human fetal hemoglobin (Hb F) have been constructed to determine what effects specific amino acid residues in the gamma chain have on the biophysical and biochemical properties of the native protein molecule. Target residues in these recombinant fetal hemoglobins were replaced with the corresponding amino acids in the beta chain of human normal adult hemoglobin (Hb A). The recombinant mutants of Hb F included rHb F (gamma 112Thr --> Cys), rHb F (gamma 130Trp --> Tyr), and rHb F (gamma 112Thr --> Cys/gamma 130Trp --> Tyr). Specifically, the importance of gamma 112Thr and gamma 130Trp to the stability of Hb F against alkaline denaturation and in the interaction with sickle cell hemoglobin (Hb S) was investigated. Contrary to expectations, these rHbs were found to be as stable against alkaline denaturation as Hb F, suggesting that the amino acid residues mentioned above are not responsible for the stability of Hb F against the alkaline denaturation as compared to that of Hb A. Sub-zero isoelectric focusing (IEF) was employed to investigate the extent of hybrid formation in equilibrium mixtures of Hb S with these hemoglobins and with several other hemoglobins in the carbon monoxy form. Equimolar mixtures of Hb A and Hb S and of Hb A(2) and Hb S indicate that 48-49% of the Hb exists as the hybrid tetramer, which is in agreement with the expected binomial distribution. Similar mixtures of Hb F and Hb S contain only 44% hybrid tetramer. The results for two of our recombinant mutants of Hb F were identical to the results for mixtures of Hb F and Hb S, while the other mutant, rHb F (gamma 130Trp --> Tyr), produced 42% hybrid tetramer. The sub-zero IEF technique discussed here is more convenient than room-temperature IEF techniques, which require Hb mixtures in the deoxy state. These recombinant mutants of Hb F were further characterized by equilibrium oxygen binding studies, which indicated no significant differences from Hb F. While these mutants of Hb F did not have tetramer-dimer dissociation properties significantly altered from those of Hb F, future mutants of Hb F may yet prove useful to the development of a gene therapy for the treatment of patients with sickle cell anemia.

Immunization with a synthetic peptide conjugate derived from the N-terminal sequence of either the beta-chain of haemoglobin or the immunosuppressive protein (reOLT 4) reduces the litter size of pregnant rats.

A synthetic peptide conjugate based on the N-terminal sequence of a 10 000 MW immunosuppressive serum protein (reOLT 4) was used to immunize female Lewis rats prior to mating, in order to determine whether blocking this protein had an effect on pregnancy. The N-terminal sequence of (reOLT 4) has close sequence homology to the beta-chain of rat haemoglobin so a peptide conjugate based on the N-terminal sequence of this protein was also used to immunize female Lewis rats. Controls included animals that were not immunized and animals that received the peptide carrier, diphtheria toxoid (DT). No statistical differences were found in gestation time or litter sizes in these groups. Differences were, however, evident between these groups and animals that received DT-(reOLT 4) (group 4) or the DT-beta-chain haemoglobin (group 5). There were no statistical differences in litter size or gestation time for group 4 when compared with group 5. Enzyme-linked immunosorbent assay and dot-blot analysis revealed that rats from both groups also had strong responses against DT, the peptide conjugate they were immunized with and the corresponding full-length protein. In both cases, animals from group 4 and group 5 had weak responses to the peptide that they did not receive, together with lower erythrocyte counts and haematocrits, and elevated heart to body weight ratios. Additionally, antibody purified on a (reOLT 4) immunoaffinity column was capable of binding to rat erythrocytes. A second investigation comparing anaemia prior to fertilization and maintained anaemia over the gestation period revealed that only the latter was capable of decreasing litter size to the same degree as obtained for groups 4 and 5. We conclude that for groups 4 and 5 it is the autoimmune effect of continual anaemia over the gestation period, mediated by autoantibodies, which results in the observed lower litter size.

2PA100 ヘモグロビンのα鎖とβ鎖のF9部位アミノ酸残基は機能的に相同か?

2PA100 ヘモグロビンのα鎖とβ鎖のF9部位アミノ酸残基は機能的に相同か?

The Hemoglobins of the Antarctic Fishes Artedidraco orianae and Pogonophryne scotti: AMINO ACID SEQUENCE, LACK OF COOPERATIVITY, AND LIGAND BINDING PROPERTIES

The oxygen-transport system of two species of Antarctic fishes belonging to the family Artedidraconidae, Artedidraco orianae and Pogonophryne scotti, was thoroughly investigated. The complete amino acid sequence of the alpha and beta chains of the single hemoglobins of the two species was established. The oxygen-binding properties were also investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes, and unstripped hemolysates. Both hemoglobins have unusually high oxygen affinity and display a relatively small Bohr effect; the Root effect is elicited only by organophosphates and is also reduced. Remarkably, the Hill coefficient is close to one in the whole pH range, indicating absence of cooperative oxygen binding which, in A. orianae hemoglobin, could be ascribed to the subunit heterogeneity shown upon oxygen dissociation. In comparison with the other families of the suborder Notothenioidei, the oxygen-transport system of these two species of Artedidraconidae has unique characteristics, which raise interesting questions on the mode of function of a multisubunit molecule and the relationship with cold adaptation.

The N-terminal Sequence Affects Distant Helix Interactions in Hemoglobin: IMPLICATIONS FOR MUTANT PROTEINS FROM STUDIES ON RECOMBINANT HEMOGLOBIN FELIX

The N-terminal 18-amino acid sequence of the beta-chain of hemoglobin, as far as the end of the A helix, has been replaced by the corresponding sequence of the gamma-chain of fetal hemoglobin with the remaining sequence of the beta-chain retained (helices B through H). The gamma-beta-chain had the correct mass, and its entire sequence was established by mass spectrometric analysis of its tryptic peptides; the alpha-chain also had the correct mass. This recombinant hemoglobin (named Hb Felix) retains cooperativity and has an oxygen affinity like that of HbA both in the presence and absence of the allosteric regulators, 2,3-diphosphoglycerate or chloride but differs from HbF in its 2,3-diphosphoglycerate response. However, Hb Felix has some features that resemble fetal hemoglobin, i.e. its significantly decreased tetramer-dimer dissociation and its circular dichroism spectrum, which measure the strength of the tetramer-dimer interface in the oxy conformation and its rearrangement to the deoxy conformation, respectively. Even though Hb Felix contains the HbA amino acids at its tetramer-dimer interface, which is located at a distance from the substitution sites, its interface properties resemble those of HbF. Therefore, the N-terminal sequence and not just those amino acids directly involved at the subunit interface contacts with alpha-chains must have a strong influence on this region of the molecule. The results reinforce the concept of fluid long range relationships among various parts of the hemoglobin tetramer (Dumoulin, A., Manning, L. R., Jenkins, W. T., Winslow, R. M., and Manning, J. M. (1997) J. Biol. Chem. 272, 31326-31332) and demonstrate the importance of the N-terminal sequence, especially in some mutant hemoglobins, in influencing its overall structure by affecting the relationship between helices.

Comparison of protein mixtures in aqueous humor by membrane preconcentration - capillary electrophoresis - mass spectrometry.

The significance of proteomic research is coupled with the recent exponential growth of these investigations. Currently, the most popular techniques used for these studies include the coupling of 1- and 2-dimensional electrophoresis with mass spectrometric analysis of the extracted and digested proteins. However, detection limits of gel staining methods have led to a search for complimentary techniques that afford the detection of lower concentrations of biologically relevant proteins. In the present studies, we have evaluated the applicability of on-line capillary electrophoresis - mass spectrometry (CE-MS) for this application. Specifically, we used membrane preconcentration-CE-MS (mPC-CE-MS) to analyze 13 samples of human aqueous humor (AH) from patients with various ocular pathologies (cataract, cataract plus glaucoma, and cataract plus pseudoexfoliation syndrome). This approach enabled rapid analysis of a relatively large volume (1 microL of each specimen, and a protein map for each was created. Measured average molecular weights (Mr) were used to tentatively identify proteins after search of the SWISS-PROT database using TagIdent from ExPaSy. Among those proteins tentatively identified are beta-2 microglobulin (Mr 11731.2), apolipoprotein A1 (Mr 28078.6) and serum albumin (Mr 66400). Proteins with Mr of 4349 (unidentified), 11731.2 (beta-2 microglobulin), 13400-14100 (immunoglobulin fragments), 28078.2 (apolipoprotein A1) and approximately 68000 (serum albumin) were observed in the majority of specimens. Generally no significant differences were noted in the protein composition of aqueous humor samples from different pathologies. However, the absence of an Mr 13345 protein and its oxidized form (Mr 13361) in samples from patients with pseudoexfoliation syndrome was noted. Occasionally the alpha-and beta-chains of hemoglobin, a contaminant in aqueous humor introduced during sampling, were also detected. We conclude from these studies that mPC-CE-MS is an attractive complimentary technique for proteome research, as this approach enables direct mapping and characterization of low concentrations of proteins that are present in complex physiologically derived fluids.

Capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for protein characterization.

On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is demonstrated for high-resolution analysis of model proteins, human hemoglobin variants, and Escherichia coli proteins. The acquisition of high-resolution mass spectra of hemoglobin beta chains allows direct identification of hemoglobin variants A and C, differing in molecular mass by 1 Da. Direct mass determination of cellular proteins separated in the CIEF capillary is achieved using their isotopic envelopes obtained from ESI-FTICR. The factors which dictate overall performance of CIEF-ESI-FTICR, including duty cycle, mass resolution, scan rate, and sensitivity, are discussed in the context of protein variants and cell lysates analyzed in this study.

Modulation of peripheral inflammation by locally administered hemorphin-7.

Sensory nerves mediate peripheral inflammation via the release of sensory peptides at the site of tissue injury. Using a blister model of inflammation, we have previously documented that endogenous opioids modulate chronic but not acute inflammation. Hemorphins are nonclassical opioid peptides found in the region of the beta-chain of hemoglobin (Hb). The heptapeptide hemorphin-7 is identical with residues 35-41 of the beta-chain of the human Hb. The aim of this study was to examine the effect of hemorphin-7 on the inflammatory response in acute and chronic injury models. We have used a vacuum-induced blister model in the footpad of anaesthetized rats to induce an inflammatory response in naive skin by (a) electrical stimulation (ES) of the distal end of the cut sciatic nerve at 20 V, 5 Hz, 2 ms for 1 min or (b) superfusion of sensory peptides; substance P (SP) or calcitonin gene related peptide (CGRP) over the blister base. In addition, we examined the effect of hemorphin-7 on the inflammatory response to SP induced in a previously injured but healed skin site (recurrent injury model) and in denervated skin site due to chronic nerve lesion (chronic injury model). The results showed that prior and concomitant perfusion of hemorphin-7 over the blister base inhibited the acute inflammatory response to ES of the sciatic nerve at C-fibre strength in a dose-dependent manner. Significant inhibition was achieved at 20 and 200 microM concentration of hemorphin-7. When hemorphin-7 (20 microM) was perfused prior to and together with SP or CGRP (both at 1 microM), over the base of acutely induced blister in naive skin, it significantly reduced the inflammatory response to SP (both plasma extravasation and vasodilatation), but was without effect on the vasodilatation response to CGRP. Naloxone, the general opioid antagonist at (1 mg/kg i.v.) reversed the inhibitory effect of hemorphin-7 on the inflammatory response to SP. On the other hand, hemorphin-7 had no effect on the inflammatory response to SP in the recurrent injury or the chronic injury models. The results of this study suggest that hemorphins might play a role in inhibiting the inflammatory response in acute, but not in recurrent or chronic injury conditions. Evidence is also provided that the modulatory inhibitory effect of hemorphin-7 is mediated via activation of opioid receptor(s). The significance of this study in conjunction with our previous work, is that it raises the possibility that different endogenous inhibitory mechanisms may operate under different injury conditions - endogenous hemorphins and opioids may modulate acute and chronic inflammation, respectively.

Leech extracellular hemoglobin: two globin strains that are akin to vertebrate hemoglobin alpha and beta chains.

Leech (Whitmania edentula, Haemadipsa zeylanica var. japonica and Erpobdella lineata) extracellular hemoglobins are basically composed of three constituent subunits, a dimer (D1 and D2 chains) and two monomers (M1 and M2 chains). We isolated these four chains from respective species by a combination of reversed-phase chromatography on a Resource RPC column and gel-filtration on a Superdex 75 column. The apparent molecular masses of the four globin chains were estimated by SDS-PAGE analysis to be 13 kDa (M1), 16 kDa (M2; 19 kDa in its reduced form) and about 27 kDa for the dimer subunit (13 kDa for D1; 15 kDa for D2), regardless of the source. The amino (N)-terminal segments (21-30 residues) from twelve globin chains of the above three species were determined and aligned. It was found that the twelve sequences could be separated into two distinct globin groups A and B. This finding supports the original idea of "two globin strains in annelid hemoglobin", which was proposed without any evidence for leech hemoglobins. Comparing the sequences in the three classes of Annelida, Hirudinea, Oligochaeta and Polychaeta, we found two invariant amino acids, Cys and Trp, which are interposed by eleven amino acid residues. Furthermore, the globin chains belonging to strain A were readily discernible as they had three more invariants, Ser-13, Asp-16 and Trp-28, while the globin chains of strain B had two more invariants, Lys-12 and Arg-27. Consequently, we propose that each of the three classes of Annelida have two distinct groups of globin chains that are akin to vertebrate hemoglobin alpha and beta chains.

Structure and Oxygen Affinity of Crystalline des-His-146β Human Hemoglobin in the T State

To correlate directly structure with function, the oxygen affinity and the three-dimensional structure of crystals of the T quaternary state of des-His-146beta human hemoglobin have been determined by polarized absorption microspectrophotometry and x-ray diffraction crystallography. In des-His-146beta, the COOH-terminal histidine residues of the beta chains of hemoglobin A have been removed. Oxygen binding to crystalline des-His hemoglobin is non-cooperative and independent of pH. The oxygen affinity is 1.7-fold greater than that of the crystalline state of hemoglobin A. Removal of His-146beta results in a small movement of the truncated COOH-terminal peptide and in a very small change in quaternary structure. Previously, similar studies on T state crystals of des-Arg-141alpha hemoglobin showed that removal of the COOH termini of the alpha chains results in much larger effects on oxygen affinity and on quaternary structure. Kinetic studies in solution reveal that at pH 7.0, the rates of CO combination with deoxygenated des-His-146beta in the absence and presence of inositol hexaphosphate are 2.5- and 1.3-fold, respectively, more rapid than for hemoglobin A. The values for des-Arg are 7.6- and 3.9-fold. The properties of the T state of hemoglobin both in the crystal and in solution are influenced to a greater degree by the interactions associated with Arg-141alpha than those associated with His-146beta.

Structural characterization of human hemoglobin crosslinked by bis(3,5-dibromosalicyl) fumarate using mass spectrometric techniques.

Diaspirin crosslinked hemoglobin (DCLHb) was analyzed by mass spectrometric-based techniques to identify the protein modifications effected by the crosslinking reaction with bis(3,5-dibromosalicyl) fumarate. DCLHb consists of two principal components. These components were isolated by size-exclusion chromatography and identified by measurement of their molecular weight using electrospray mass spectrometry and subsequent peptide mass mapping and mass spectrometric sequence analysis of their individual digests. Three major RP-HPLC fractions were observed from the major hemoglobin in DCLHb. Their MWs matched the MW of heme, intact hemoglobin beta-chain, and two hemoglobin alpha-chains crosslinked by a fumarate moiety, respectively. The minor HPLC peaks of DCLHb were also separated, and characterized by mass spectrometric methods. These minor components revealed additional details of the structural nature of covalent modification of DCLHb.

Effect of different proteases on bitterness of hemoglobin hydrolysates.

Hemoglobin was hydrolyzed by several enzymes (Proctase, Alcalase, Neutrase, papain). Hydrolysates were analyzed (degree of hydrolysis, gel permeation on Superose 12 column, tasting) and fractionated by ultrafiltration and 2-butanol extraction. The bitter peptides were isolated and identified. The results were compared with those already obtained with peptic hemoglobin hydrolysates. All the findings were confirmed. Ultrafiltration concentrated bitter compounds in the fraction corresponding to 500-5000 Da, and these compounds were selectively extracted by 2-butanol. All the bitter peptides belonged to the same fragment of the beta-chain of bovine hemoglobin. Finally, the use of a Superose 12 chromatographic column for easy detection of bitter hydrolysates without sensory analysis could be generalized for hemoglobin hydrolysates.

Use of a polybrene capillary coating in capillary electrophoresis for rapid analysis of hemoglobin variants with on-line detection via an ion trap storage/reflectron time-of-flight mass spectrometer.

A polybrene capillary coating in capillary electrophoresis (CE) has been used for rapid analysis of hemoglobin variant digests. The use of the polybrene capillary coating has allowed sufficient separation to resolve the large number of digest products formed upon tryptic digestion of the whole protein, so that prior separation of the hemoglobin alpha and beta chains is not required. The resolution of the digest peaks obtained by CE is sufficient so that even single amino acid substitutions can easily be detected using UV absorption detection. The digest is further analyzed by capillary electrophoresis separation with on-line detection using electrospray ionization interfaced to the ion trap storage/reflectron time of flight device (CE/ESI-IT/reTOF), where a comparison of the total ion electropherograms and mass spectra of the mutant and normal hemoglobins can detect the presence of a mutation site. The CE separation and mass analysis can be accomplished in typically 10-15 min. The unique capability of the CE/ESI-IT/reTOF system for detection of fast separations with narrow peaks that may be under 1 s fwhm is demonstrated. The speed of this system is essential for resolution of the large number of peaks that are separated in a short time duration using CE separations.