Sintesi e studio spettroscopico mediante NMR di sequenze omologhe di peptidi bioattivi derivanti dalla proteolisi dell'emoglobina (emorfine)

The hemolysate of the Antarctic teleost Gobionotothen gibberifrons (family Nototheniidae) contains two hemoglobins (Hb 1 and Hb 2). The concentration of Hb 2 (15-20% of the total hemoglobin content) is higher than that found in most cold-adapted Notothenioidei. Unlike the other Antarctic species so far examined having two hemoglobins, Hb 1 and Hb 2 do not have globin chains in common. Therefore this hemoglobin system is made of four globins (two alpha- and two beta-chains). The complete amino-acid sequence of the two hemoglobins (Hb 1, alpha2(1)beta2(1); Hb 2, alpha2(2)beta2(2)) has been established. The two hemoglobins have different functional properties. Hb 2 has lower oxygen affinity than Hb 1, and higher sensitivity to the modulatory effect of organophosphates. They also differ thermodynamically, as shown by the effects on the oxygen-binding properties brought about by temperature variations. The oxygen-transport system of G. gibberifrons, with two functionally distinct hemoglobins, suggests that the two components may have distinct physiological roles, in relation with life style and the environmental conditions which the fish may have to face. The unique features of the oxygen-transport system of this species are reflected in the phylogeny of the hemoglobin amino-acid sequences, which are intermediate between those of other fish of the family Nototheniidae and of species of the more advanced family Bathydraconidae.

The Conger conger (conger eel) haemoglobin (Hb) system is made of three components, one of which, the so-called cathodic Hb, representing approx. 20% of the total pigment, has been purified and characterized from both a structural and functional point of view. Stripped Hb showed a reverse Bohr effect, high oxygen affinity and slightly low cooperativity in the absence of any effector. Addition of saturating GTP strongly influences the pH dependence of the oxygen affinity, since the reverse Bohr effect, observed under stripped conditions, is converted into a small normal Bohr effect. A further investigation of the GTP effect on oxygen affinity, carried out by fitting its titration curve, demonstrated the presence of two independent binding sites. Therefore, on the basis of the amino acid sequence of the alpha- and beta-chains, which have been determined, a computer modelling study has been performed. The data suggest that C. conger cathodic Hb may bind organic phosphates at two distinct binding sites located along the central cavity of the tetramer by hydrogen bonds and/or electrostatic interactions with amino acid residues of both chains, which have been identified. Among these residues, the two Lys-á(G6) (where the letter refers to the haemoglobin helix and the number to the amino acid position in the helix) appear to have a key role in the GTP movement from the external binding region to the internal central cavity of the tetrameric molecule.

In this note we describe two new equine hemoglobin phenotypes found during a survey of the Murgese horse, a rare Apulian native breed, among whose ancestors the Arabian surely plays an important role. To date we have analysed about 300 individual hemolysates by different chromatographic analyses (PAGIF, IPG, CMC). The results pointed out two unusu- al patterns where the ratio of the ?24Phe60Gln band to the ?24Phe60Lys band was 93:7 and 70:30 rather than 60:40 which would have been expected of BII homozygote. Given that the three horses exhibiting the unusual patterns shared a common ancestor and that none of the possible combinations of the known haplotypes can account for 7-8% ?24Phe60Lys, reasonably a triplicated arrangement has to be postulated.

The blood of Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) is completely devoid of hemoglobin. Icefishes have developed compensatory adaptations that reduce oxygen demand and enhance oxygen transport. Oxygen delivery to tissues occurs by carrying the gas physically dissolved in the plasma. To evaluate the evolutionary pathway leading to the icefish hemoglobinless phenotype, the adult and embryonic/juvenile gene complexes from a closely related, red-blooded notothenioid species were isolated and characterized. The hybridization pattern of notothenioid adult globin cDNAs showed that the genomes of three icefish species retain transcriptionally inactive alpha1-globin-related DNA sequences, which are identical truncated variants of the alpha1-globin gene of the red-blooded fish, containing part of intron 2, all of exon 3, and the 3'-untranslated region. The icefish genomes have no beta-globin genes. Furthermore, Southern blots of genomic DNA from red- and white-blooded (two species) notothenioids, probed with fragments of the genes flanking the ends of the embryonic/juvenile complex, indicated that icefishes have also lost embryonic/juvenile globin genes. It is proposed that inability to express hemoglobin arose from a single, large-scale deletional event, which removed all icefish globin genes with the exception of the 3' end of alpha1.

The Arctic fish Anarhichas minor, a benthic sedentary species, displays high emoglobin multiplicity. The three major hemoglobins (Hb 1, Hb 2, and Hb 3) show important functional differences in pH and organophosphate regulation, subunit cooperativity, and response of oxygen binding to temperature. Hb 1 and Hb 2 display a low, effector-enhanced Bohr effect and no Root effect. In contrast, Hb 3 displays pronounced Bohr and Root effects, accompanied by strong organophosphate regulation. Hb 1 has the beta (beta(1)) chain in common with Hb 2; Hb 3 and Hb 2 share the alpha (alpha(2)) chain. The amino acid sequences have been established. Several substitutions in crucial positions were observed, such as Cys in place of C-terminal His in the beta(1) chain of Hb 1 and Hb 2. In Hb 3, Val E11 of the beta(2) chain is replaced by Ile. Homology modeling revealed an unusual structure of the Hb 3 binding site of inositol hexakisphoshate. Phylogenetic analysis indicated that only Hb 2 displays higher overall similarity with the major Antarctic hemoglobins. The oxygen transport system of A. minor differs remarkably from those of Antarctic Notothenioidei, indicating distinct evolutionary pathways in the regulatory mechanisms of the fish respiratory system in the two polar environments.

Tetrameric hemoglobins are the most widely used systems in studying protein cooperativity. Allosteric effects in hemoglobins arise from the switch between a relaxed (R) state and a tense (T) state occurring upon oxygen release. Here we report the 2.0-A crystal structure of the main hemoglobin component of the Antarctic fish Trematomus newnesi, in a partial hemichrome form. The two alpha-subunit iron atoms are bound to a CO molecule, whereas in the beta subunits the distal histidine residue is the sixth ligand of the heme iron. This structure, a tetrameric hemoglobin in the hemichrome state, demonstrates that the iron coordination by the distal histidine, usually associated with denaturing states, may be tolerated in a native-like hemoglobin structure. In addition, several features of the tertiary and quaternary organization of this structure are intermediate between the R and T states and agree well with the R --> T transition state properties obtained by spectroscopic and kinetic techniques. The analysis of this structure provides a detailed pathway of heme-heme communication and it indicates that the plasticity of the beta heme pocket plays a role in the R --> T transition of tetrameric hemoglobins.

The erythroid-specific enhancer within hypersensitivity site 2 (HS2) of the human beta-globin locus control region is required for high level globin gene expression. We used an oligonucleotide of the NF-E2 tandem repeat, within HS2, as recognition site probe to screen a K562 cDNA library for interacting transcription factors. A 2.3 kb full length cDNA encoding the b-zip transcription factor MafF was isolated. MafF can form both homodimers and high affinity heterodimers with Nrf1, Nrf2 and Nf-E2, three members of the CNC-bZip family. Despite obvious structural similarities with the other small Maf proteins, MafF differs in its tissue distribution and its inability to repress transcription when overexpressed as homodimer. In fact, in different cell lines and on different promoters (beta-globin, gamma-globin and glutamylcysteine synthetase genes) the MafF homodimers do not appreciably affect transcription of target promoters, whereas MafF/CNC member heterodimers act as weak transcriptional activators. Even though MafF was cloned using probes derived from the globin LCR, it is in the context of the GCSl promoter and in combination with Jun that MafF shows a rather distinct and specific regulatory role. These observations suggest that a complex network of small Maf and CNC?AP1 protein interactions might be involved in regulating transcription in diverse tissues or developmental stages.

Hemoglobin function is modulated by several non-heme ligands; among these effectors, organic phosphates generally bind to heterotropic sites with a one-to-one stoichiometry. The phosphate binding site of human hemoglobin is located at the interface between the two beta chains. An additional binding site for polyanions has been studied at the molecular level (Tamburrini, M., A. Riccio, M. Romano, B. Giardina, and G. di Prisco. 2000. Eur. J. Biochem. 267:6089-6098) in the hemoglobins of the south polar skua (Catharacta maccormicki). It is formed by a cluster of six positive charges of both alpha chains (Val-1, Lys-99, Arg-141); the two Lys-99alpha have an essential role in the site structure. The present investigation, carried out on skua deoxyhemoglobins by using a molecular dynamics approach, confirms the structural feasibility of the additional site, possibly having the role of an entry-leaving site, and leads to the proposal of a novel migration pathway for phosphate along the central cavity of hemoglobin from one binding site to the other, occurring according to the hypothesis of a site-site migratory mechanism, which may assign a functional role to the central cavity. The role of Lys-99alpha was further confirmed by molecular dynamics experiments on the mutant Lys-99alpha-->Ala in which, at the end of the simulation, the phosphate was external to the additional site.

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.