Bovine hemoglobin alpha-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 a-globin variants to which the extensive mass spectrometric analysis attributed a molecular mass of 15026.47 +/- 0.44 Da and 15079.86 +/- 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 ex genes of several mammals, we conceive that the alpha(89His-->Tyr) is an allelic form of the (I) alpha gene while the alpha(131Asa-->Ser) is an allelic form of the (II)alpha gene. ((C) Societe francaise de biochimie et biologie moleculaire/Elsevier, Paris).