 Reference Base  Molecular basis for ATP/2,3-Bisphosphoglycerate control s... |
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Abbasi, A.; Weber, R.E.; Braunitzer, G.; Goeltenboth, R., 1987. Molecular basis for ATP/2,3-Bisphosphoglycerate control switch-over (poikilotherm/homeotherm): an intermediate amino-acid sequence in the hemoglobin of the Great Indian Rhinoceros (Rhinoceros unicornis, Perissodactyla). Biological Chemistry Hoppe Zeyler 368: 323-332, figs. 1-7, tables 1-4
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Physiology |
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Indian Rhino |
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Hemoglobin is the oxygen carrier in the blood of all vertebrates except for a few antarctic deepsea fishes. Its oxygen-binding efficiency is regulated by allosteric cofactors like GTP (mainly found in fish), ATP (mainly found in fish, amphibians and reptiles), IPP (mainly in birds) and DPG (mainly in mammals). Studies on the primary structure of hemoglobin and its interaction with these anionic organic effectors have led to the identification of positively charged key amino-acid residues involved in the regulation. Thus, while DPG as well as ATP/GTP bind at position ?I Val and ?82 Lys, DPG binds to ?2 His and ?143His of mammalian hemoglobin molecules, whereas the nucleoside triphosphates specifically interact with ?2 Glu/Asp and 143 Arg of fish hemoglobin.
Some members of the mammalian order Perissodactyla (families Rhinocerotidae and Tapiri- dae) are unique in having glutamic acid in position ?2. one of the binding sites for ATP/GTP. We have extended our studies on the Rhinocerotidae and report the primary structures of the two hemoglobin components from Rhinoceros unicornis, which show aspartic or glutamic acid, respectively, at position ?2 and present oxygenation properties and the sensitivities to DPG and ATP for the two hemoglobin components. The different allosteric control mechanisms in homeotherms and poikilothenns are examined on the basis of the new sequence data.
Material and Method
Preparation of hemolysate and electropheresis
Hemolysate was prepared as described earlier. The number of hemoglobin components was determined by polyacrylamide gel electrophoresis using 7.5% gels and Tris/glycine buffer, pH 9.5 (Fig. la). The number of polypeptide subunits was determined in the presence of 8M urea and Triton X- 100 according to Niter et al.(fig. 1B).
Separation of Hemoglobin components.
Whole hemolysate was subjected to ion exchange chromatography in a column (1.6 x 15 cm) of DEAE-Sephacel (Pharmacia, preswollen), equilibrated with 50 mM Tris/HCI buffer pH 8.5. Elution was carried out with a gradient of 0.02- 0.2 M NaCI. Flow rate was maintained at 21 ml/h and fractions of 5 ml each were collected. The absorbance was monitored at 415 and 280 nm.
Separation of globin chains
A globin sample reduced with dithiothreitol for 3 h under nitrogen was applied to a 1.6 x 15-cm column of carboxymethyl cellulose (Whatman CM-52) equilibrated with 8M urea containing 0.2% mercapto-ethanol and 25 mM sodium acetate pH 5.7. A salt gradient of 0.02-0.08M NaCl was employed for eluting the polypeptides. Purity of the subunits was examined by polyacrylamide gel electrophoresis in the presence of urea and Triton X-100 (Fig. lb).
Enzymatic cleavage
Native or oxidized polypeptide chains were digested with TPCK-treated trypsin.(Worthington) at pH 10.7 and 9.5 for 3 h end an enzyme to substrate ratio of 5:100. The hydrolysate was then titrated to pH 4.5, centrifuged and subjected to gel filtration.
Fractionation of peptides
The enzymatic hydrolysate was prefractionated on Sephadex G-25 fine (2.6 x 140 cm) equilibrated and eluted with 0.1M acetic acid at a flow rate of 21 ml/h. Individual peaks were then rechromatographed by high-performance liquid chromatography on Lichrosorb RP-2 using an ammonium acetate/acetronile system.
Chemical cleavage
Hydrolytic cleavage of the Asp-Pro bond was performed to obtain the C-terminal part of the polypeptide. The sample was dissolved in 6M guanidinium hydrochloride/70'% formic acid and maintained at 42?C for 55 h. The hydrolysis products were chromatographed on Sephadex G-50 fine (2.6 x 160 cm) with 8M urea in 10% formic arid. The C-terminal peptide was desalted over Sephadex G-25 in 1 M acetic acid.
Amino-acid analysis
Peptides were hydrolysed in constant boiling HCI (5.7M) at 110?C for 20 h. Cysteine and methionine were estimated after performic acid oxidation. Tryptophan was estimated after hydrolysis in the presence of 6% thioglycollic acid. The analyses were performed on a Bio- tronic autoanalyser LC 5000.
Sequence analysis
Sequence studies were carried out by automatic degradation of cryptic and hydrolytic cleavage products in the liquid phase sequencers 890B & 890f (Beckman Instruments), Intact polypeptide or small lysine peptides were sequenced with Quadrol (0.25 and 1.0 M) after coupling with reagent I or reagent IV, whereas 3-diethylamino propyne was employed in the case of arginine peptides and the C-terminal prolyl-peptide. Conversion of the thiazolinone to phenyl-thiohydantoin was performed with 3m trifluoroacetic acid at 80?C. Identification of the phenylthiohydantoin derivatives of amino acids was followed either on thin-layer pre-coated silica gel plates (E. Merck 11151 or by high-performance liquid chromatography.
Oxygen equilibrium measurements
Solutions of the two major components showing some oxidation were reduced by addition of a trace of sodium dithionite, saturation with carbon monoxide and dialysis against several changes of CO-saturated, 0.05M Tris buffer containing 5 x 10-4 m EDTA. The hemoglobin solutions were concentrated by pressure dialysis through a molecular sieve membrane with cut-off at 10 kDa (nucleopore Corp.. Pleasanton, California).
Oxygen equilibria were determined using a modified oxygen diffusion chamber technique, in which oxygen tensions in the gas mixtures passing through the chamber were increased stepwise using Westhoff gas mixing pumps (D-4630 Bochum) to mix air and pure nitrogen (99.998%). The equilibria were determined at 37 and 25?C at different pH values (obtained by buffering to 0.1M with Na Hepes, in the presence of 0.1 M KCl and in the absence or presence of DPG, ATP and GTP. The concentrations of these organic phosphates in the stock solution used were assayed enzymatically using Boehringer test chemicals
Results
Polyacrylamide gel electrophoresis of the hemolysate showed the presence of two components, A and B, occuring in a ratio of 4:6. Electrophoresis of the hemolysate under dissociating conditions revealed three polypeptide subunits ( Fig. 1).
DEAE-Sephacel chromatography of the hemolysate resulted in the isolation of the two components in pure form (Fig. 2). Separation of the crude globin on CM-cellulose in the presence of HM urea revealed four well-defined peaks (Fig. 3a), whereas only three peaks were identified on high-performance liquid chromatography of the hemolysate with Nucleosil C-4 using 0.1% tritluoroacetic acid and acetonitrile gradients (Fig. 3b). All four peaks from CM-cellulose chromatography were therefore analysed. Tryptic peptides were prefractionated on Sephadex G-25 and then chromatographed on Lichrosorb RP-2 with 50mM ammonium acetate and 0-40% acetonitrile gradients ( Fig. 4).
Sequence studies on the intact polypeptides resulted in the identification of the first 42 N- terminal residues. Tryptic peptides revealed the position of the remaining amino acids. Their amino-acid compositions are presented in Tables 2-4 (see supplementary material). Over- lapping sequence was provided by the hydrolytic C-terminal prolyl-peptide. The complete amino-acid sequence of the - and -chains is presented in Fig. 5. Alignment of the sequence with human hemoglobin leads to 18 exchanges in the case of - and 22 exchanges each for the ?I and II subunits requiring a minimum of 21 nucleotide substitutions (3 of which arise from two-point mutations) for the -chain and 27 nucleotide substitutions for I and ?II (5 of these as a result of two point mutations). These substitutions alter the 1?1 subunit cooperativity at 4 points, i.e. at ?55 Met to Leu, ?112 Cys to Val, ? 116 His to Gln, ? 125 Pro to Glu, 1?2 contact at 43 Glu to Asp, and haem contacts at ?70 Ala to Ser and ?85 Phe to Tyr. In addition to this important DPG binding site ? NA2 His has been mutated to Asp or Glu, respectively. Within the two ?-chains three differences were 1ocated at ?NA2 Asp/Glu, ?A3 Glu/Gly and ?GH3 Gln/Lys.
Figs. 6 and 7 show the effect of DGP, ATP and GTP on the hemoglobin oxygenation proper- ties of the isolated Rhinoceros hemoglobin components. At pH 7.4 and 37?C components A and B display similar P50 values (13.8 and 12.0 mmHg) and the Bohr factors (= Ig P50/pH = -0.60 and -0.58 respectively). At 25 ?C the P50 values for HbA and HbB were 6.3 and 6.0. whereas were -0.72 and -0.67 respectively. These data reflect values for the overall heats of oxygenation of approximately - 46 and -42 kJ/mol O2 (about -11 and -10 kcal/moll for components A and B, respectively. The inverse relationship between and temperature accords with findings for human hemoglobin and shows a qualitatively similar relationship between temperature and the pK values for ionization in the Rhinoceros hemoglobin. At pH 7.0 to 7.5 which may he expected
to encompass physiological conditions, the cooperativity coefficient, n was about 2.0.
Both hemoglobins showed only slight sensitivity to organic phosphates at physiological pH conditions. Accordingly, DPG, ATP and GTP increased the 1g P50 of stripped Hb A by only 0.05 (Fig, 6). Component B, however, exhibited slightly greater sensitivities to DPG ( 1g P50 approximately 0.06 and 0.13 for ATP and DPG respectively (Fig. 7).
Discussion
Rhinocerotidae together with Tapiridae (tapirs) and Equidae (horses) constitute the order Perissodactyla, which was one of the richest orders in the tertiary period some 65 million years ago. Today the order is restricted to six species of horses, four species of tapirs and five species of rhinoceros. Studies on the order Perissodactyla have been of particular interest owing to the fact that their fossil record is well documented and have contributed much to present-day understanding of mammalian evolution.
Studies on the various members of this order aimed at unravelling the primary structure of hemoglobins undertaken by Braunitzer and colleagues show the presence of glutamine at ?N?2 in the case of equidae and glutamic acid in the case of Tapiridae and Rhinocerotidae. Since interaction of ?NA2 His with DPG in mammals is well documented, these results evoked much interest as to whether these sequences reflect an intermediate mechanism that might have occured during the course of evolution. ATP is the most effective organic modulator of oxygen affinity in fishes, amphibians and reptiles and a stereochemical model for its interaction has been proposed by Perutz and Brunori. Table 1 shows the various sites of its interaction. The hemoglobin of the Great Indian Rhinoceros evidences an intermediate control.
With regard to oxygen binding the two hemoglobin components exhibit similar characters. The half-saturation oxygen tensions for HbA and HbB (17.8 and 15.8 mmHg, respectively at pH 7.2) correspond accurately with that (17.1 mmHg) given at this pH for the whole hemo- lysate from the White-Mouthed Rhinoceros (Ceratotherium simum) by Baumann and co-workers, despite the fact that their measurements were carried out at about 8 times greater hemoglobin concentrations. This indicates that molecular dissociation did not influence the oxygen affinities recorded here. In contrast to the findings that ATP and DPG had the same effect on the P50 at 20-fold molar excess over hemoglobin tetramers in Ceratotherium, our data show a greater DPG than ATP effect in the most abundant Rhinoceros component (HbB) at saturating cofactor concentrations (cf. Fig. 7).
Although the two components are structurally different with ?I NA2 Asp, A3 Glu, GH3 Gln compared to ?II NA2 Gfu, A3 Gly, 6113 Lys a 'division of labour' such as that observed in some teleost hemolysates, where the cationic component exhibits higher affinities, smaller Bohr factors and different temperature effects compared to the anionic component is not seen. The sensitivities of the Rhinoceros hemoglobins to phosphates are low, with the values of 1g P50 (the difference between values in the absence and presence of the phosphates) equalling 0.05-0.13, compared to the values of about 0.4-0.6 found for fish hemoglobins with ATP and mammalian hemoglobins with DPG. This indicates that the substitution of NA2 His in human hemoglobin A for ?NA2 Asp or Glu in rhinoceros drastically reduces DPG binding, while nucleoside triphosphate binding is also hindered by the persistence of the other DPG sites.
The temperature effect (as expressed in terms of the overall heat of oxygenation, H) is simi- lar to that recorded in other mammals, despite the higher variable body temperature (between 30 and 42 ?C, P.G. Wright, personal communication) in the Rhinoceros, particularly in its extremities.
Given the monophyletic origin of mammals, the occurence in Rhinoceros hemoglobins of amino-acid residues that are involved in nucleoside-triphosphate binding in homeotherm vertebrates, cannot be considered an evolutionary relict, whereby it must reflect a reversal of evolution in terms of oxygen affinity control The question of the adaptive significance of such secondary development in Rhinocerotidae and Tapiridae remains speculative.
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