Hb rainier [β145(HC2)Tyr→Cys] in Italy. Characterization of the amino acid substitution and the DNA mutation

Abstract

A high oxygen affinity hemoglobin variant was identified in a 53-year-old male patient from Napoli (Italy), suffering from pulmonary thromboembolism and polycythemia. A detailed structural characterization of the variant hemoglobin was carried out, both at the protein and DNA levels, by a combination of DNA sequencing and allele-specific amplification techniques with mass spectrometric procedures. The amino acid substitution was found to be Tyr-->Cys, and the corresponding DNA mutation was identified as A-->G at the second position of codon 145 of the beta chain. These variations indicated the presence of Hb Rainier. Haplotype analysis of DNA polymorphisms showed that the beta-globin gene from Hb Rainier was associated with haplotype II. Moreover, structural analyses provided direct identification of an intramolecular disulphide bridge joining the newly inserted beta145Cys with beta93Cys. This is the first report of the occurrence of Hb Rainier in Italy.

Polymerization of hemoglobins in Arctic fish: Lycodes reticulatus and Gadus morhua

“…Several S-S bridges were formed by C-terminal Cys b146 CH3, indicating higher reactivity, and/or high flexibility of the domain where this residue is located. This behavior is similar to that of the human variant Hb Rainier (30), and of C. kumu Hb (23), where Cys b49 CD8 replaces His, commonly found in other fish Hbs. Hb polymerization has also been recorded in other teleosts (33).…”

Characterization of the elusive disulfide bridge forming human Hb variant: Hb Ta-Li β83 (EF7)Gly → Cys by electrospray mass spectrometry

“…The mass difference of 1 Da per chain from the expected mass change is explained by inter-molecular -S-S-bond formation where each variant ␤ subunit loses one hydrogen atom from the newly introduced Cys residue. If there had been an intra-molecular -S-S-bridge, the expected mass difference per chain would be 2 Da, which has been observed by others in the monomer Hb Rainier [13]. This Ta-Li example illustrates how limiting the deconvolution output mass range can give misleading results when there are dimers in the sample, a situation that can be remedied by reprocessing the data to accommodate dimers or by reducing the sample.…”

“…In Hb Pô rto Alegre (␤9Ser 3 Cys), the first described variant involving Cys, the Cys residue is present on the external surface of the Hb tetramer and therefore forms inter-molecular -S-S-bridges [12]. Hb Rainier (␤145Tyr 3 Cys), the most extensively studied Cys containing variant, involves the internal ␣ 1 ␤ 2 contact of the Hb molecule and forms intra-molecular -S-S-bridges with ␤93Cys [13]. On the other hand, in Hb Nunobiki (␣141Arg 3 Cys) where the variant substitution is at the C-terminal end (internal ␣ 1 ␤ 2 contact), no intra-or inter-molecular -S-S-bridges were observed [14].…”

Hb J-CAPE TOWN [α92(FG4)Arg→Gln (α1), CGG→CAG] in Southern Italy Found in a Patient with Erythrocytosis

“…Individual globin chains were purified by reversed phase HPLC on an HP 1100 system (Agilent Technologies) essentially as described by Carbone et al (9). The variant globin was digested with trypsin and the resulting peptide mixture was directly analyzed by matrix-assisted laser desorption ionization (MALDI) MS using a Voyager DE PRO Reflectron instrument (Applied BioSystems, Framingham, MA, USA) as reported before (10). The peptide mixture was fractionated by reversed phase HPLC on an HP 1100 system (Agilent Technologies) using a C 18 narrow bore column (11).…”

“…The variant chain was purified by preparative HPLC, digested with trypsin and an aliquot of the resulting peptide mixture was directly analyzed by MALDI/MS, as previously described (10). An anomalous peak, absent in the normal α chain tryptic digest, was detected at m/z 1059.8 and tentatively assigned to the abnormal peptide 91-99 with a molecular mass decreased by 28 Da (αT-8,9, expected mass value 1087.7 Da), thus confining the structural variation within this region.…”