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The primary structure of the hemocyanin Aa6 subunit form the scorpion Androctonus australis was resolved by using protein sequencing and mass spectrometry for analysis of the polypeptide chain and of fragments obtained by CNBr, trypsin, and chymotrypsin cleavage. Due to the high sensitivity of the methodologies used, only a small amount of material, less than 1 mg, was consumed. The complete sequence is composed of 626 amino acid residues and the protein is not glycosylated but probably phorylated at Ser374. Its molecular mass measured by mass spectrometry (71890 ±7 Da) is about 30Da higher than the mass calculated from the sequence data (71860.1 Da). The origin of this difference is not clear but could result from minor molecular heterogeneities. Within the chelicerates, the Aa6 subunit of the arachnid A. australis shares 405 identical residues with chain e of another arachnid. Eurypelma californicum and 399 with chain α of the merostom Tachypleus tridentatus. The degrees of identity between these three subunits, which are known to occupy the same location in the native hemocyanin oligomers, are significantly higher than those existing between the subunits a,d, and e of E. californicum. This favors the hypothesis that gene duplications, leading to separate chains in one species, have occurred before the divergence between arachnids and merostoms. [ABSTRACT FROM AUTHOR]

The elemental content of the iron cores of haemosiderins isolated from animal and human tissues has been determined to ascertain whether changes in composition are correlated with structural differences previously identified in these mineralisation products. Significant differences were observed in the elemental composition of haemosiderins isolated from patients subjected to desferrioxamine-chelation therapy compared to patients who had been venesected. The P/Fe molar ratio was considerably higher in haemosiderin isolated from treated primary haemochromatosis (0.83), compared to untreated primary haemochromatosis (0.10) and treated secondary haemochromatosis (0.25), and this could account for the amorphous nature of these iron cores. The levels of M/Fe (M = Ca, Cu, Zn) were reduced in the haemosiderins derived from treated secondary haemochromatosis patients, possibly due to the chelation of these ions by desferrioxamine therapy. In an experimentally iron-loaded rat, receiving either desferrioxamine or 1,2-diethyl-3-hydroxypyrid-4-one, selective decreases in these three elements were also observed after two weeks of desferrioxamine therapy. Such changes may be important determinants in the modification of biomineralisation of the iron cores. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the hemocyanin subunit c from the spiny lobster, Panulirus interruptus, has been determined. The elucidation was mainly based on three digests, with CNBr, trypsin and endoproteinase Glu-C, respectively. Additional evidence was obtained by sequencing of peptides from an endoproteinase Lys-C digest. Subunit c is a polypeptide with 661 amino acid residues and with a carbohydrate group attached to residue 476 in the third domain. No heterogeneity was observed. The degree of identity with subunit a is 59%. Some differences with subunit a are an N-terminal extension of six residues, a one-residue C-terminal extension, and a three-residue deletion. Furthermore, carbohydrate attachment is in a different position, as are most half-cystine residues. Limited trypsinolysis resulted in cleavage at the same site as in subunits a and b. [ABSTRACT FROM AUTHOR]

Flash photolysis kinetics of carbon monoxide hemoglobin show a decrease in the fraction of ligand recombination occurring as geminate when the hemoglobin has fewer ligands bound. Fully saturated samples, normally referred to as R state, show approximately 50% geminate phase, while samples at low saturation (T state) show less than 3%. The latter result was obtained by photolysis of samples with a short delay after stopped flow of solutions of deoxy hemoglobin (Hb) and ligand. The decrease in the fraction of geminate phase was also observed using a double flash technique. The transient mixture of R and T states generated by flash photolysis of Hb-CO was probed with a weaker time-delayed photolysis pulse. The kinetics of both the geminate and bimolecular phases following the second pulse were measured. The fraction geminate signal was least at delays where the maximum proportion of liganded T state tetramer is expected. The biphasic bimolecular process is also an indicator of the allosteric state of Hb. The populations of R and T may be determined from the overall ligand recombination kinetics; however, the analysis is model-dependent. The fraction geminate reaction may provide a rapid measure of the amount of liganded hemes in the R and T states. [ABSTRACT FROM AUTHOR]

The structural change of lobster hemocyanin in cooperative O2 binding was studied by the dye-binding method. It was found that neutral red shows an O2-1inked binding to hemocyanin with a higher affinity for the oxy form. The number of the dye-binding sites was estimated to be three in the hexameric molecule of oxyhemocyanin. The course of the structural change in the partially oxygenated hemocyanin was examined using the absorbance change of the bound dye as a measure. It was found that the fractional change in the dye binding was considerably greater than the degree of O2 saturation of hemocyanin. The three-state allosteric model, which was proposed for explanation of the O2 binding properties of lobster hemocyanin [N. Makino (1986) Eur. J. Biochem. 154, 49- 55], was also consistent with the effects of the dye on the O2 binding to the native hemocyanin. On the basis of this model, the dye binding to partially oxygenated hemocyanin could be connected with the populations of the affinity states. It was inferred that the binding of neutral red reflects the quaternary structure of the protein. In contrast, O2 binding to the stripped (EDTA-treated) hemocyanin showed a considerable decrease in the cooperativity in the presence of the dye. The O2-binding isotherms could not be explained by the three-state model. It is suggested that the subunit interaction is partially blocked by the dye in the absence of divalent cations. [ABSTRACT FROM AUTHOR]

Hemocyanin from the freshwater snail Lymnaea stagnalis is high-molecular-mass copper-containing oxygentransport protein, which occurs freely dissolved in the memolymph. It is a glycoprotein containing fucose, xylose, 3-O-methylgalactose, mannose, galactose, N-acetylglucosamine residues as sugar constituents. The N-glycosidic carbohydrate chains of this glycoprotein were released by hydrazonolysis of a pronase digest and subsequently fractionated as oligosaccharide-alditols on Bio-Gel P-4 followed by Lichrosorb-NH2. Investigation with 500-MHz 1H-NMR spectroscopy, in conjunction with sugar and methylation analysis revealed the lowest-molecular-mass glycan chain to have the structure:[this is equation can not converted in ASCII text]. [ABSTRACT FROM AUTHOR]

Reports on the isolation of hemocyanin mRNA from tarantula hemocytes in its in vitro translation. Technique for obtaining transcription into cDNA and cloning of the cDNA; Characterization of the clone characterized by restriction mapping and sequencing.

The reaction of Limulus polyphemus hemocyanin with a dye, bromthymol blue, was examined by equilibrium dialysis, spectrophotometric titration and stopped-flow methods. Oxy-hemocyanin contained one binding site per hexamer unit. The dye binding was linked to oxygenation, and the affinity of the dye for the oxy form was about 10 times as high as that for the deoxy form. Conversely, the dye increased the O2 affinity of hemocyanin. Hemocyanin showed a simple hyperbolic binding curve in the bromthymol blue titration, whereas the time course of the reaction was generally biphasic. It was inferred from the kinetic analyses that the reaction proceeds in two steps. The first bimolecular step is characterized by an increase in the apparent pKa of the bound dye, while the second unimolecular step by a red shift of the absorption band of the unionized dye. The dye binding to partially oxygenated hemocyanin was examined spectrophotometrically; the fractional change in the binding was found to be ahead of the increase in the average degree of O2 saturation. It was concluded that the structural changes in hemocyanin which lead to the increased dye affinity take place at an early stage of the ligand binding sequence. [ABSTRACT FROM AUTHOR]

Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of keyhole limpet henocyanin (KLH: from Megathura crenulata. A prosobranch gastropod) gave a molecular mass of kDa for the subunit of KLH1 and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/Page. Within the 400-kDa KLH1 subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45-60 kDa functional domains (termed 1a through 1h) and determined their sequential arrangement. The KLH1 domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C-terminal domain 1h, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLH1 and KLH2 (8.3 Mda versus 7.5 Mda), correlating with 20 copies of a financial copies to a functional 1h domain. It was also shown that the KLH1 didecamer can be rapidly split (minutes) into a almost homogeneous population of stable decamers by increasing the pH of the Tris/saline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl2 and 5mM MgCl2, to pH 8.5. reformation of the didecamers occurred more slowly (days) upon analysis against the pH7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously observed KLH1 multidecamers, which could be structurally distinguished from the established multidecamers of KLH2. [ABSTRACT FROM AUTHOR]

The mode of action towards oligopeptides and proteins of hydrolase H purified from rabbit skeletal muscle was studied. The presence of protamine or α-N-benzoylarginine p-nitroanilide (an endopeptidase substrate) chart both the Km and V values of the enzyme towards Leu-β-napthylamide (an aminopeptidase substrate). This indicates that the binding site for an endopeptidase substrate is different from that for an aminopeptidase substrate. Hydrolase H as an aminopeptidase displayed broad specificity. The enzyme hydrolyzed various dipeptides readily except the dipeptides containing Pro or an amino acid with a hydrophobic β-branched chain at the NH2 terminus. Pro and Val at the NH2 terminus of tripeptides were also difficult to release, whereas Ile and Val of tetrapeptides were easily released in contrast with those of dipeptides. The longer the peptide chain of Glyn (n = 2, 3, 4), the more susceptible was it to hydrolase H. Hydrolase H behaved as an endopeptidase only towards protamine among the proteins tested. The other proteins, casein, bovine serum albumin, myofibrils, troponin, hemoglobin, sarcoplasmic proteins, and myoglobin were probably attacked only by the aminopeptidase activity of the enzyme. [ABSTRACT FROM AUTHOR]

The quaternary structure of the (4 × 6)-mer hemocyanin from the scorpion Androctonus australis previously published [Lamy. J., Bijiholt. M. M. C. Sizaret, P.-Y., Lamy. J., and van Bruggen. E. F. J. (1981) Biochemistry, 20, 1849-1856] has been refined. The relative positions in the half molecule of subunits Ac 3A and Ac 38 compared to those of Aa 3C and Aa 58 have been established by double labeling of the (2 × 6)-mer with binary mixtures of subunit-specific Fab fragments. The results show that subunits Ac, 38 and Ac, 58 are located in the same hexamer while Aa 3A and Au 3C are in the other half of the (2 × 6)-mer. The choice of the enantiomer was deduced from a careful examination of' electron micrographs of the native molecule. Finally a position was assigned to each of the 24 subunits on the flip and flop faces as defined by Van Heel and Frank [Ultramicroscops, 6, 187-194 (1981)]. [ABSTRACT FROM AUTHOR]

In subjects carrying the haemoglobin Hasharon mutation (α47 Asp ↵ His), originally from the delta of the Po river (Northern Italy), the concentration of the α-globin variant has been evaluated and found to be approximately 32%, a value definitely higher than that reported for the same mutant haemoglobin in other regions. Restriction enzyme analysis has-been carried out on the DNA from these subjects; the data obtained indicate the presence of three α-globin genes per diploid cell. Family studies further show that the two normal genes are located on one chromosome and the Hasharon gene on the other. The origin of the single a-gene in the Hasharon-carrying subjects of the Ferrara region is discussed in connection with their haematological and biosynthetic data. [ABSTRACT FROM AUTHOR]

A Southern Italian patient homozygous for hemoglobin Lepore disease synthesizes approximately 3 % Lepore δβ-globin chains (relative to α chains) in the reticulocytes. Measurement of β-like RNA sequences by hybridization to complementary DNA specific for β-globin demonstrates a low level (1-2% relative to a sequences) of these sequences in cytoplasmic RNA from reticulocytes or spleen cells, suggesting that the Lepore gene is expressed into mRNA at a lower extent than normal α or β genes; the comparison with the level of β-like sequences found in nuclear RNA (6 - 8 %) further supports this conclusion and indicates, in addition, that Lepore RNA might be degraded at a faster rate than normal, 2-3% β-like sequences are found in nuclear RNA in three cases of homozygous β0-thalassemia, setting the highest possible estimate for the δ-RNA level; this figure suggests that the `δ-promoter'-dependent Lepore δβ gene is somehow more actively expressed than the δ gene. [ABSTRACT FROM AUTHOR]

The binding of l-anilino-8-naphthalene sulfonate (ansyl) to native and copper-free hemocyanin of Octopus vulgaris has been studied in different conditions by measuring the fluorescence properties of the probe in the presence of hemocyanin. Native hemocyanin, either in the oxygenated or in the deoxygenated state, does not bind ansyl. The binding of ansyl with apohemocyanin induces a strong increase (from 0.004 to 0.6-0.7) of the quantum yield and a blue shift from 520 nm to 460 nm of the emission maximum indicating the presence of ansyl binding sites in the protein. Experimental evidence is reported that the binding occurs at the copper-binding site of the protein. The dissociation constants of the ansyl-hemocyanin complexes are equal to about 10-4 M, i.e. they are of the same order of those obtained with other proteins. The number of binding sites (n) of apohemocyanin for ansyl depends on the conformational state of the protein and ranges from 0.15 -0.80 mol/mol protein (Mr, 50000), depending on pH, ionic strength, and urea concentration. A negative interaction between the ansyl binding sites has been suggested. [ABSTRACT FROM AUTHOR]

The effect of external parameters on the relaxation process of the hemochrome-type non-equilibrium conformation in partially reduced methemoglobin has been investigated. The relaxation of the intermediate ferrous low-spin state to the high-spin equilibrium conformation of hemoglobin appears to be facilitated particularly by protons and phosphate ions. In addition to studying the spin-state transition in aquomethemoglobin we have also studied it in complexes of the heme group in methemoglobin with fluoride, azide and cyanide anions. [ABSTRACT FROM AUTHOR]

Optical difference spectrophotometry has been applied for measuring the early part of the O2 dissociation curve of human hemoglobin (Hb); i.e., below 1% O2 saturation levels or tog [y(1-y)] from -3.0 to -2.0, where y = fractional O2 saturation. Two matched cuvettes, sealed on glass tonometers of known volume, were filled with the same deoxygenated Hb solution (65 μM in tetramer) under atmospheric pressure of pure argon. The pO2 values were calculated after the addition to the sample tonometer of accurate volumes of O2/argon mixtures the composition of which was varied by using a precise gas-mixing pump. 4–5 points of O2 saturation below y = 0.01 were thus measured, which allowed for the calculation of A1, the first association constant of O2 to the tetrameric Hb. We have compared the effects of protons, chloride ions, CO2 and bisphospho-glycerate (P2-glycerate) on the pH dependence (alkaline Bohr effect) of A1 and p50. At low chloride concentrations (0.005 M) the alkaline Bohr effect at A1 was identical to that measured at p50, indicating that exactly one-fourth of the Bohr protons were released at the first oxygenation step. At higher chloride concentration (0.15 M) A1 was decreased and p50 was increased as was the Bohr effect. CO2 additions (pCO2 38 Torr, 5050 Pa, at I = 0.15 M) lead to a decreased A1 and increased p50 but to a large decrease of the alkaline Bohr effect. The effect of CO2 was predominant at A1, indicating a large release of carbamino adducts from the tetrameric Hb at the first step of ligation. P2-glycerate decreased At and increased p50 values. The alkaline Bohr effect was enhanced at p50 by P2-glycerate but lowered at A1. These results are compared to those already published by other workers. The variations of the pH dependence of A1 in the presence of effectors indicate that A1 should be considered as a phenomenological parameter refering to one of the possible structures that the Hb tetramer can take in the unliganded state. They indicate also that the whole Hb molecule feels the first oxygenation step, which itself implies structural changes within the quaternary structure. [ABSTRACT FROM AUTHOR]

The kinetics of carbon monoxide binding following fast reduction of the valency hybrids α2+β2coand α2coβ2+ by hydrated electrons have been studied at different degrees of reduction. The results show that at pH 6.0 and 7.0 reduction of one heme group yields a species which reacts fast with carbon monoxide (rate constant of the order of 106 M-1 s-1. At pH 6.0 the intermediates α2coβ2 and α2β2co bind carbon monoxide with a rate characteristic of the T state. At pH 7.0 α2coβ2co is for the greater part in the T state, while in the case of α2β2co the R and the T state are about equally populated. [ABSTRACT FROM AUTHOR]

Haemoglobins of Artemia salina were investigated during development and the results obtained are summarised as follows. a) A new haemoglobin (haemoglobin N) was induced de novo in the hatching nauplius, but not in the prenauplius nor in the embryo. b) Haemoglobin N seemed not to be synthesised in the adult. c) The adult contained three distinct haemoglobins (haemoglobins I, II and III) in a quantitative ratio of 1:6:3, respectively, and these haemoglobins were not detected in nauplius. d) Isoelectric points determined were 5.7, 5.6, 5.7, and 5.9 for haemoglobin N, I, II and III, respectively. e) All four haemoglobins contained one large polypeptide (Mr = 1.1 – 1.3 × 105) and possibly two minor polypeptides (Mr = 8–9.5 × 104 and Mr = 5.4 × 104, respectively). [ABSTRACT FROM AUTHOR]

Binding of adenosine triphosphate to human methaemoglobin was investigated at pH 6.2 and 7.2 by the use of an ultracentrifugation method. Two classes of binding sites regarding their affinity were found. The association constant of the class with lower affinity will be diminished by temperature elevation from 5° to 21°. The heat of binding which was evaluated to be -6.8 kcal/ mote supports the hypothesis derived from previous experiments on pH dependence of binding, that imidazole groups might be involved. [ABSTRACT FROM AUTHOR]

A new Lepore-type hemoglobin in a person of Negro descent has been identified. In this person a minor hemoglobin (Hb-LeporeBaltimore) was detected. An abnormal δβ-chain was isolated and fingerprinted. This hybrid-type protein presumably arose as a consequence of unequal crossing over between the δ and β genes, as was concluded for Hb-LeporeBoston and Hb-LeporeHollandia. In the new Lepore-type hemoglobin described here the crossover could be placed between residues δ50 and β86. This type of crossover should occur with the highest frequency if crossing over is equally frequent between any nucleotide and if selection favours none of the crossover types. [ABSTRACT FROM AUTHOR]

A new Lepore-type hemoglobin in a person of Negro descent has been identified. In this person a minor hemoglobin (Hb-LeporeBaltimore) was detected. An abnormal δβ-chain was isolated and fingerprinted. This hybrid-type protein presumably arose as a consequence of unequal crossing over between the δ and β genes, as was concluded for Hb-LeporeBoston and Hb-LeporeHollandia. In the new Lepore-type hemoglobin described here the crossover could be placed between residues δ50 and β86. This type of crossover should occur with the highest frequency if crossing over is equally frequent between any nucleotide and if selection favours none of the crossover types. [ABSTRACT FROM AUTHOR]