Separation of glycosylated haemoglobins using immobilized phenylboronic acid. Effect of ligand concentration, column operating conditions, and comparison with ion-exchange and isoelectric-focusing

Haemoglobins from diabetic and non-diabetic individuals have been separated by affinity chromatography using immobilized phenylboronate, which interacts specifically with diol-containing compounds such as glycosylated haemoglobin. The effects of ligand concentration, flow rate, column geometry, preincubation of sample, buffer composition and temperature have been investigated. Significant correlation was found between results from affinity-chromatography and ion-exchange and isoelectric-focusing methods. Isoelectric-focusing of the haemoglobin fractions obtained from affinity chromatography indicate that, in addition to haemoglobin A1c, some haemoglobin A is also bound to immobilized phenylboronic acid. Assays of haemolysates obtained from red blood cells incubated in glucose solutions suggest that unstable pre-(haemoglobin A1c) does not interfere. The assay is not affected by the presence of haemoglobin F.     

A partial amino acid sequence for sheep haemoglobin A

Amino acid analysis and terminal-group analysis of tryptic and chymotryptic peptides from sheep haemoglobin A have enabled a partial amino acid sequence to be worked out. By comparing this partial sequence with the known amino acid sequences of human haemoglobins A and F as well as horse slow haemoglobin the most probable sequence of sheep haemoglobin has been deduced.     

Amino acid sequence of the beta-chain of the tetrameric haemoglobin of the bivalve mollusc, Anadara trapezia

The amino acid sequence of the beta-chain of the principal haemoglobin from A. trapezia has been determined. The sequence was deduced from the sequences of tryptic peptides, which were fractionated using highperformance liquid chromatography and peptide mapping. Additional sequence data, particularly for the large tryptic peptides, was obtained from enzyme digests of both cyanogen bromide fragments and large citraconyltryptic peptides. The beta-chain has 151 residues which is longer than all the other sequenced haemoglobin chains except the alpha-chain of A. trapezia, which is 153 residues in length. The residues corresponding to those normally in the D helix are absent in this beta-chain. The additional residues are contributed by an extension of the N-terminal region, which was also found to be acetylated. Comparison of the beta-chain amino acid sequence with that of the alpha-chain of A. trapezia, the dimeric chain of A. trapezia, and the dimeric chain of A. broughtonii showed 53% identity in each case. In the E and F helices, the homology is particularly noticeable. There is 100% homology in the F helix of all four chains. The dimeric globin of A. trapezia also shows 100% homology with the beta-chain in the E helix, while the alpha-chain shows 75%. If the tertiary structure of the alpha- and beta-chains of A. trapezia haemoglobin is the same as that of horse haemoglobin, then there are many changes in the alpha 1 and beta 2 contact site residues.     

Temperature- and pH-dependence of the oxygen-binding reaction of human fetal haemoglobin.

O2 binding to human haemoglobin F0 was studied at high haem concentrations (3 mM) in the temperature range 15-35 degrees C and in the pH range 6.8-8.7 at 25 degrees C. Comparison with O2 binding to human adult haemoglobin A0 under identical solution conditions reveals striking similarities in the Bohr effect and the enthalpy of oxygenation between the two haemoglobins.     

Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident.     

Macromolecular isoforms of Daphnia magna haemoglobin

The haemoglobin (Hb) of Daphnia magna acclimated to different oxygen conditions was sampled, and in its natively assembled state it was separated by chromatofocusing. The Hb isoforms were analysed for their subunit composition under denaturating conditions by two-dimensional gel electrophoresis. The Hb system is suggested to consist of three predominant Hb aggregates, which are characterised by a specific subunit composition and synthesised in response to different ambient oxygen conditions. In normoxia, a dominant Hb aggregate (DmHbI) with a pI of 4.4-4.6 was composed of subunits B, C, E, F and G. In severe hypoxia, a different dominant Hb isoform (DmHbIII) with a pI of 5.7-5.9 was composed of subunits A, B, C, D, E and F. Further analyses in moderate hypoxia provided evidence for a third Hb isoform (DmHbII) composed of subunits B, C, D, E and F. Sequence alignment and homology modelling of the tertiary structure of the D. magna Hb domains 1 and 2 revealed functionally relevant substitutions of amino acid residues at positions B10, E7 and E11, which determine the functional properties of D. magna haemoglobin in terms of haem contact, oxygen binding and affinity. Both domains are predicted to possess the common haemoglobin fold, but helices C and D are not properly formed, and helix G is interrupted by a short coil.     

Haemoglobin Saki alpha 2 beta 2 14 Leu-Pro(a11) structure and function.

The characterization of haemoglobin Saki alpha 2 beta 2 14 Leu-Pro(a11) is described. This new mutation is unique since it only induces modification of the stability of the molecule. In vitro precipitation of haemoglobin Saki upon heat or in the presence of chemicals is compared to the stability of haemoglobin A and haemoglobin S.     

Improved survival in homozygous sickle cell disease: lessons from a cohort study.

OBJECTIVE: To examine whether simple interventions in a sickle cell clinic improve survival in sickle cell disease. DESIGN: Survival curve analysis and hazard ratios in a cohort study followed from birth. SETTING: MRC Laboratories (Jamaica) at the University of the West Indies, and Victoria Jubilee Hospital, Kingston, Jamaica. SUBJECTS: 315 patients with homozygous sickle cell disease detected during the screening of 100,000 consecutive non-operative deliveries between June 1973 and December 1981 at the main government maternity hospital, Kingston, Jamaica. INTERVENTIONS: Prophylactic penicillin to prevent pneumococcal septicaemia, parental education in early diagnosis of acute splenic sequestration, close monitoring in sickle cell clinic. MAIN OUTCOME MEASURES: Survival. RESULTS: Survival appeared to improve, the log rank test for trend comparing the first, second, and last third of the study reaching borderline significance (P = 0.05). Combined deaths from acute splenic sequestration and pneumococcal septicaemia-meningitis declined significantly (test for trend, P = 0.02). CONCLUSION: Early diagnosis and simple prophylactic measures significantly reduce deaths associated with homozygous sickle cell disease.     

The thermal denaturation of human oxyhaemoglobins A, A2, C and S

1. The time-courses of thermal denaturation of human oxyhaemoglobins A, A(2), C and S at 45 degrees C were studied by following the increase in protein fluorescence. Haemoglobins S and C were less stable than haemoglobin A, whereas haemoglobin A(2) was considerably more stable. 2. The time-courses of denaturation did not follow first-order kinetics and could be fitted most simply to a co-operative scheme in which the partial denaturation of the alpha chain preceded that of the beta chain. 3. The denaturation of these haemoglobins was studied as a function of temperature by using optical rotatory dispersion. Haemoglobin A(2) was again more stable than the others. The addition of small quantities of haemoglobin A(2) had a disproportionate effect on the stability of haemoglobin C. 4. The thermodynamic parameters of the denaturation process were calculated.     

Structure-function relationships in the low-affinity mutant haemoglobin Aalborg (Gly74 (E18)beta----Arg).

Haemoglobin Aalborg (Gly74 (E18)beta----Arg) has a reduced oxygen affinity, in both the absence and the presence of organic phosphates; it has a raised affinity for organic phosphates, and it is moderately unstable. By contrast, haemoglobin Shepherds Bush (Gly74 (E18)beta----Asp) has an increased oxygen affinity in both the absence and the presence of organic phosphates, a diminished affinity for organic phosphates and is also unstable. We have determined the crystal structure of deoxyhaemoglobin Aalborg at 2.8 A resolution and compared it to the structures of deoxy- and oxyhaemoglobin A and of deoxyhaemoglobin Shepherds Bush. The guanidinium group of Arg74(E18)beta protrudes from the haem pocket and donates hydrogen bonds to the E and F helices. The carboxylate group of Asp74(E18)beta forms a hydrogen bond only with residue EF6 and is partially buried, which may be why haemoglobin Shepherds Bush appears to be more unstable than haemoglobin Aalborg. To discover why the latter has a low oxygen affinity, we superimposed the B, G and H helices of haemoglobin A, whose conformation is known to be unaffected by ligand binding, on those of haemoglobin Aalborg. This also brought helices E and the haems into superposition, but revealed a shift of the F helix of deoxyhaemoglobin Aalborg towards the EF-corner. This shift is opposite to that which occurs on ligand binding and on transition to the quaternary oxy-structure, and is linked to an increased tilt of the proximal histidine residue away from the haem axis. Since the relative positions of helices E and F and of the haem group are thought to be the main determinants of the changes in oxygen affinity, the shift of helix F may account for the reduced oxygen affinity of haemoglobin Aalborg. The shift may be due to a combination of steric and electrostatic effects introduced by the arginine residue's side-chain. The effects of the arginine and aspartate substitutions at position E18 beta on the 2,3-diphosphoglycerate affinity are equal and opposite. They can be quantitatively accounted for by the electrostatic attraction or repulsion by the oppositely charged side-chains.     

Evidence for contrasting modes of selection at interacting globin genes in the European rabbit (Oryctolagus cuniculus)

In hybrid zones between genetically differentiated populations, variation in locus-specific rates of introgression may reflect adaptation to different environments or adaptation to different genetic backgrounds. The European rabbit, Oryctolagus cuniculus, is well-suited to studies of such hybrid zone dynamics because it is composed of two genetically divergent subspecies that hybridize in a zone of secondary contact in central Iberia. A species-wide survey of allozyme variation revealed a broad range of locus-specific divergence levels (F(ST) ranged from 0 to 0.54, mean F(ST)=0.16). Interestingly, the two loci that fell at opposite ends of the distribution of F(ST) values, haemoglobin alpha-chain (HBA) and haemoglobin beta-chain (HBB), encode interacting subunits of the haemoglobin protein. The contrasting patterns of spatial variation at these two loci could not be reconciled under a neutral model of population structure. The HBA gene exhibited higher-than-expected levels of population differentiation, consistent with a history of spatially varying selection. The HBB gene exhibited lower-than-expected levels of population differentiation, consistent with some form of spatially uniform selection. Patterns of linkage disequilibrium and allele frequency variation do not appear to fit any simple model of two-locus epistatic selection.     

Haemoglobin Hiroshima and the Mechanism of the Alkaline Bohr Effect

HAEMOGLOBIN Hiroshima is a variant with interesting physiological properties^1,2 discovered in a Japanese family. Its Bohr effect is halved, its oxygen affinity at physiological pH increased about three-fold and haem-haem interaction is somewhat reduced compared with normal haemoglobin. In 0.1 M NaCl solutions initially stripped of phosphate, 2,3-diphospho-glycerate (2,3-DPG) diminishes the oxygen affinity as in haemoglobin A (H. F. Bunn, unpublished results). The amino-acid substitution originally deduced for this abnormal haemoglobin was histidine 143 (H21)β ? aspartic acid¹. It was possible to conceive of a mechanism which accounted for its diminished Bohr effect³, but the normal response of its oxygen affinity to 2,3-DPG was inconsistent with the proposed role of histidine 143 in 2,3-DPG binding by haemoglobin A^4,5. An X-ray crystallographic study of deoxyhaemoglobin Hiroshima has now revealed that the replacement occurs not in position 143 but 146β. This was confirmed by chemical methods and the physiological properties of this haemoglobin are now satisfactorily accounted for. The results support the role of histidine 146β in the alkaline Bohr effect⁶.     

Calcium plays an important role in the regulation of hepatic branched-chain 2-oxo acid dehydrogenase activity

A fetal haemoglobin variant was noted in a healthy Jamaican infant of mixed African/Chinese extraction. A two-dimensional chromatogram of the soluble tryptic peptides (Tp) showed 2 ‘new’ ones/ One was composed of the last 4 residues of the usually insoluble Tpγ41–59. To permit a tryptic split this required a change of residue γ55 Met to Lys or Arg. The other new Tp contained arginine and was in the position expected for a Tpγ41–55 (55 Arg). As the material was limited it could not be analysed. When after more than 6 years no example of Hb F Kingston had become available it was decided to describe the variant on the basis of the present evidence.     

Water hyacinth decline across Lake Victoria—Was it caused by climatic perturbation or biological control? A reply

Several authors have identified that a combination of interacting factors involving the El Niño of 1997/1998 and biocontrol by weevils resulted in the rapid reduction of water hyacinth across Lake Victoria [Albright, T.P., Moorhouse, T.G., McNabb, J., 2004. The rise and fall of water hyacinth in Lake Victoria and the Kagera River Basin 1989–2001. J. Aquat. Plant Manage. 42, 73–84; Williams, A.E., Duthie, H.C., Hecky, R.E., 2005. Water hyacinth in Lake Victoria: why did it vanish so quickly and will it return? Aquat. Bot. 81, 300–314; Wilson, J.R.U., Ajuonu, O., Center, T.D., Hill, M.P., Julien, M.H., Katagira, F.F., Neuenschwander, P., Njoka, S.W., Ogwang, J., Reeder, R.H., Van, T., 2007. The decline of water hyacinth on Lake Victoria was due to biological control by Neochetina spp. Aquat. Bot. 87, 90–93]. It would appear to us that any disagreement between these papers centres on the order and magnitude of the contributory factors. In this reply we reiterate that whilst weevils almost certainly played their part, the synchronous lake wide reduction of water hyacinth during the second quarter of 1998 was the result of the 1997/1998 El Niño.     

Haemoglobin Arya: alpha 2-47 (CD5), aspartic acid yields asparagine.

A new haemoglobin variant (haemoglobin Arya), is described from an Iranian female. The substitution is at residue 47 (CD5) of the alpha chain in which aspartic acid has been substituted by asparagine. The presence of haemoglobin Arya was not associated with clinical symptoms. This variant has normal stability at 50 degrees C, but is slightly unstable when tested at 55 degrees C.     

The relationship between normocytic, hypochromic anaemia and iron concentration together with hepatic enzyme activities in cattle infected with Fasciola hepatica

Erythrograms determined from whole blood analyses and serum analyses for aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) activities, and iron concentration, were used in infected and uninfected cattle to determine the type of anaemia and degree of hepatic damage caused by Fasciola hepatica. Blood samples from 86 infected and 30 uninfected cattle were taken at slaughter. Haematological analyses revealed decreased levels of packed cell volume (PCV), haemoglobin concentration, mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in infected compared with uninfected cattle (P < 0.05). A decrease in the concentration of serum iron was also observed in infected cattle compared with uninfected cattle (P < 0.05). Significant increases in AST, GGT and ALP activities were observed in cattle infected with F. hepatica when compared with uninfected cattle (P < 0.05). It was concluded that the anaemia observed in cattle infected with F. hepatica is a normocytic, hypochromic anaemia and the most important aetiology of the anaemia is the chronic blood loss due to the blood-sucking activity of the adult flukes and leakage of blood from the bile duct to the intestine, which results in iron deficiency. The increased activities of serum enzymes indicated chronic hepatic and bile duct injuries associated with chronic infection with F. hepatica.     

Clinical Studies and Physiological Properties of Hopkins-2 Haemoglobin

HAEMOGLOBIN Hopkins-2 (Ho-2) was discovered in a family in which haemoglobin S was also present¹. Independent segregation of the two abnormal haemoglobins provided the first convincing evidence that two genetic loci are concerned with synthesis of the haemoglobin molecule^1,2. Subsequently, evidence was obtained that the abnormality in haemoglobin Ho-2 is in the alpha chain³, whereas the lesion in haemoglobin S is in the beta chain of globin. We have reported the structural abnormality in Ho-2 and we now present a revised pedigree and the clinical status of carriers; we attempt to relate these findings to functional abnormalities exhibited by the haemoglobin.     

Clinical Studies and Physiological Properties of Hopkins-2 Haemoglobin

HAEMOGLOBIN Hopkins-2 (Ho-2) was discovered in a family in which haemoglobin S was also present¹. Independent segregation of the two abnormal haemoglobins provided the first convincing evidence that two genetic loci are concerned with synthesis of the haemoglobin molecule^1,2. Subsequently, evidence was obtained that the abnormality in haemoglobin Ho-2 is in the alpha chain³, whereas the lesion in haemoglobin S is in the beta chain of globin. We have reported the structural abnormality in Ho-2 and we now present a revised pedigree and the clinical status of carriers; we attempt to relate these findings to functional abnormalities exhibited by the haemoglobin.     

REVIEWS

The Chemistry of Plant Processes . By C. P. W hittingham
Physics in Botany . By J. A. R ichardson
The Structure and Life of Bryophytes . By E. V. W atson
Third Congress of the European Association for Research on Plant Breeding
Textbook of Pollen Analysis . By K. F aegri and J. I versen
Patterns in the Balance of Nature and Related Problems in Quantitative Ecology . By C. B. W illiams
Grasses and Grasslands . Edited by C. B arnard
The Ecology of Rocky Shores . By J. R. L ewis
The Living Plant . By A lan B rook
The Biological Control of Insect Pests and Weeds . Edited by P aul D e B ach assisted by E vert I. S chlinger
British Ecological Society Jubilee Symposium . Edited by A. M acfadyen and P. J. N ewbould     

Electron addition to the (FeO2) unit of oxyhaemoglobin Glycera

Exposure of glassy solutions containing the monomeric fraction of the oxyhaemoglobin of the polychaete annelid Glycera dibranchiata to 60Co gamma-rays at 77 K resulted in electron addition to the (FeO2) moiety. The form of the g tensor components obtained from the ESR spectrum indicates that the spin-density on oxygen is much greater than that observed for similar paramagnetic centres formed in haemoglobin A or myoglobin. A major difference between these monomer haem units and normal haem units is that the distal histidine (E7 58) is replaced by leucine. We therefore postulate that the oxygen in the (FeO2)- units formed in haemoglobin A and myoglobin is hydrogen-bonded to the NH group of the distal histidine, whilst that of the (FeO2)- units in haemoglobin Glycera are not hydrogen-bonded. However, on annealing to approx. 160 K the spectrum changed irreversibly into one resembling those for (FeO2)- units in haemoglobin A and myoglobin. We postulate that this is caused by hydrogen-bonding to a water molecule in the haem pocket. Exposure of the polymeric fractions of haemoglobin Glycera to gamma-rays gave an (FeO2)- unit with an ESR spectrum remarkably similar to that obtained from oxymyoglobin. The X-ray structure of this protein is unknown but we suggest that our results could indicate the presence of a distal histidine in this material.