文摘
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 α-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 α-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 α genes of several mammals, we conceive that the α89 His→Tyr is an allelic form of the Iα gene while the α131 Asn→Ser is an allelic form of the IIα gene.
