L. Ferrara

Evidence for the presence of two different β-globin chains in the hemoglobin of the river buffalo (Bubalus bubalis L.)

1. Hemoglobin (Hb) of the river buffalo (Bubalus bubalis L.) was studied by employing isoelectric focusing (IEF) in the 6.7-7.7 pH range and by IEF in ultra-narrow immobilized pH gradient (IPG) 7.1-7.5. 2. Three Hb BB phenotypes were identified which were characterized by sets of two or four Hbs with different isoelectric points. 3. These type were called BB, BsBs and BBs, the Bs phenotype showing Hbs with slightly slower mobility. 4. Analysis of constituent globin chains by acid urea polyacrylamide gel electrophoresis in the presence of Triton X-100, provided clear evidence of a novel polymorphism at the beta-globin level. 5. Titration curves of beta-globins from heterozygous Hb BBs indicated a single curve thus suggesting the absence of a net charge in all the pH field. 6. A neutral-to-neutral amino acid replacement probably differentiates the two beta-globins.

Bovine hemoglobin α-globin chain polymorphism: Primary structure determination of two new genetic variants by mass spectrometry and amino acid sequencing

The present work describes the biochemical procedures used to identify the cause of a quantitative and qualitative hemoglobin polymorphism found in Podolian cattle. First, to analyze the different phenotypes, isoelectric focusing (IEF) of hemoglobins and RP-HPLC of globin chains was carried out; secondly, to determine accurately the globin molecular masses, electrospray mass spectrometry was performed and finally to check the entire amino acid sequences of the proteins, several enzymatic digests were analyzed by fast atom bombardment mass spectrometry (FAB-MS) and Edman degradation procedure. As to the qualitative polymorphism, the results of RP-HPLC show the presence of two alpha-globin variants to which the extensive mass spectrometric analysis attributed a molecular mass of 15,026.47 +/- 0.44 Da and 15,079.86 +/- 0.66 Da and whose respective primary structure differed from that of the common alpha-globin chain in the amino acid substitution Asn-->Ser at position 131 and the other in the replacement of the histidine residue at position 89 with tyrosine. As to the quantitative polymorphism, on the basis of the expression gradient found out in the duplicated alpha genes of several mammals, we conceive that the alpha 89 His-->Tyr is an allelic form of the I alpha gene while the alpha 131Asn-->Ser is an allelic form of the II alpha gene.

Electrophoretic and chromatographic evidence for allelic polymorphisms in the river buffalo α-globin gene complex

Isoelectric focusing in the ultranarrow immobilized (7.1–7.5) pH gradient (IPG) of hemoglobin and high-performances liquid chromatography (HPLC) of globin chains were used to investigate Hb polymorphism in Italian river buffalo. Six different phenotypes, each characterized by two or four different Hbs, were detected by IPG, whereas two differentIIα-globin chains were separated from two differentIα-chains by HPLC. Two α-chains (Iα1 andIIα3), and Hbs with similar mobilities (Hb1 andHb3), were associated with the AA Hb phenotype: two α-chains (Iα2 andIIα4), and Hbs with different mobilities (Hb2 andHb4), were associated with the BB phenotype: two sets of doublet Hbs were associated with the AB phenotype, thus suggesting allelic polymorphisms at the two α loci. An allele at the β locus is responsible for increasing to as many as eight the number of different Hbs, thus further complicating the notable Hb polymorphism of the river buffalo.

Water buffalo (Bubalus bubalis) hemoglobins: an electrophoretic and chromatographic study

1. Hemoglobins from three phenotypes of Italian water buffalo (Bubalus bubalis), named AA, AB and BB, were selected by starch gel electrophoresis at alkaline pH and analyzed using polyacrylamide gel isoelectric focusing and subsequent analysis of titration curves to reveal differences between two types of hemoglobin identified as Hb fast and Hb slow. 2. Globins from Hb fast and Hb slow were purified by fast protein liquid chromatography (FPLC). Electrophoretic differences were found in the respective alpha-chains using polyacrylamide gel disc-electrophoresis at acid pH, polyacrylamide gel isoelectric focusing and by subsequently analyzing titration curves. 3. The results suggest that the alpha chains of Hb fast and Hb slow, called I alpha and II alpha, respectively, differ in at least two aminoacid residues. Subsequently, these amino acids were identified as lysine and cysteine.

FAB overlapping: a strategy for sequencing homologous proteins

Abstract Extensive similarity has been shown to exist between the primary structures of closely related proteins from different species, the only differences being restricted to a few amino acid variations. A new mass spectrometric procedure, which has been called FAB-overlapping, has been developed for sequencing highly homologous proteins based on the detection of these small differences as compared with a known protein used as a reference. Several complementary peptide maps are constructed using fast atom bombardment mass spectrometry (FAB-MS) analysis of different proteolytic digests of the unknown protein and the mass values are related to those expected on the basis of the sequence of the reference protein. The mass signals exhibiting unusual mass values identify those regions where variations have taken place; fine location of the mutations can be obtained by coupling simple protein chemistry methodologies with FAB-MS. Using the FAB-overlapping procedure, it was possible to determine the sequence of α 1 , α 3 and β globins from water buffalo ( Bubalus bubalis hemoglobins (phenotype AA). Two amino acid substitutions were detected in the buffalo β chain (Lys16 → His and Asn118 → His) whereas the α 1 chains were found the α 1 and α 3 chains were found to contain four amino acid replacements, three of which were identical (Glu23 → Asp, Glu71 → Gly, Phe117 → Cys), and the insertion of an alanine residue in position 124. The only differences between α 1 and α 3 globins were identified in the C -terminal region; α 1 contains a Phe residue at position 130 whereas α 3 shows serine at position 132.

NUCLEOTIDE SEQUENCE OF A CDNA CODING FOR BOVINE MITOCHONDRIAL ASPARTATE AMINOTRANSFERASE

Aspartate aminotransferase is a pyridoxal-phosphate dependent enzyme which plays a key role in cell metabolism. We describe the cloning and sequence analysis of the cDNA encoding bovine mitochondrial aspartate aminotransferase and compare the sequence with those of isoenzymes from other mammalian species. An adult bovine heart cDNA library constructed in lambda lambda gt11 was screened using two 32P-end labeled synthetic oligonucleotides. From the screening of the cDNA library two positive clones were isolated. A subclone in pEMBL18, 6B2, generated from the longest recombinant phage was further analyzed. This clone contains an insert of 2500 bp with an Open Reading Frame of 1287 bp that encodes a protein of 430 amino acids. The deduced amino acid sequence confirms previous results obtained by mass spectrometric sequencing. We calculated the percentage of amino acid identity for each protein pair and for each comparison the average number of amino acid substitution per site (Kaa); the lowest Kaa values were obtained from the comparison between the bovine and pig enzymes. This study shows that the rate of evolution of mammalian mitochondrial AspAT is lower and more constant than the equivalent cytosolic enzyme and adds to the growing body of knowledge on the evolution of the aspartate aminotransferase.

Cloning and sequence analysis of a cdna encoding bovine cytosolic aspartate aminotransferase

1. Complementary DNA encoding cytosolic aspartate aminotransferase was isolated from an adult bovine heart library. 2. The amino acid sequence deduced for the protein (412 amino acids) is extremely similar (> 94% identity) to that of porcine cytosolic aspartate aminotransferase but interesting differences were noticed comparing the position of cysteine residues.

Electrospray mass spectrometric analysis of river buffalo (Bubalus bubalis) hemoglobins. Re-examination of α1 and α3 globin chain sequences

Abstract 1. 1. The accurate molecular weight of the globin chains from river buffalo hemoglobin components has been determined by means of electrospray mass spectrometry. 2. 2. The ES/MS analysis demonstrated that all the buffalo Hb components share a common β chain whose mass value coincides with that expected on the basis of the reported sequence. 3. 3. The AA phenotype α 1 and α 3 globin chains exhibited a 30 Da mass difference as compared to their predicted molecular weights. Careful re-examination of the two α globin sequences by FAB/MS revealed the occurrence of sequence errors and hence the correct primary structure of both α chains was established.

Phenotyping hemoglobin polymorphisms of sheep

Abstract Phenotyping hemoglobin variants of a flock of adult Altamurana sheep led to individuals with an unusual level of polymorphism being found. To find the best hemoglobin band resolution, isoelectric focusing (IEF) in traditional and in immobilized pH gradients (IPGs) of six different hemoglobin phenotypes were compared. Such a comparison afforded the following results: (1) all hemoglobin variants focus in a range of 0.7 pH units (6.7–7.4); (2) traditional IEF in the presence of chemical spacers allows the resolution of α 8Ala hemoglobin which usually remains undetected; (3) owing to the high number of hemoglobin variants in the samples, IPGs were performed in a range of 1 pH unit and exhibited almost the same results as IEF in the presence of chemical spacers. In conclusion with traditional IEF procedures, chemical spacers provided improved resolution of hemoglobin variants and the same results as IPG gels in 1 pH unit.

Crystallization and preliminary X-ray analysis of the river buffalo (Bubalus bubalis L.) BB phenotype carbonmonoxyhaemoglobin.

Ruminant haemoglobin (Hb) extracted from river buffalo (Bubalus bubalis) has been purified and crystallized. Two different Hb forms of the phenotype BB gave isomorphous crystals which diffracted to 2.8 A resolution and were not sensitive to radiation damage. Crystals of CO Hb have space group P2(1)2(1)2(1) with unit-cell parameters a = 54.8, b = 64.0, c = 158.6 A, and contain one Hb molecule per asymmetric unit.