Préparation et caractérisation physico‐chimique partielle de la transferrine sérique ovine

Abstract

Preparation and Partial Physico‐Chemical Characterization of Sheep‐Serum Transferrin Sheep‐serum transferrin shows marked polymorphism and more than 20 alleles have been identified although only 4 or 5 of these have a frequency higher than 1%. Each of the alleles has two bands in starch‐gel electrophoresis, corresponding to a major and a minor fraction. This paper describes the isolation and partial characterisation of the two fractions from the transferrin of sheep homozygous for Tf B. The purification consisted of: (a) precipitation by ammonium sulphate, (b) chromatography on CM‐cellulose and, finally (c) chromatography on DEAE‐Sephadex. The purification procedure had no effect on the electrophoretic mobility of the two fractions and they appeared homogeneous by starch‐gel and polyamide‐gel electrophoresis and by immuno‐electrophoresis. The amino‐acid compositions of both fractions were very similar and the sequences of the first eight amino‐acid residues: Ser‐Pro‐Glu‐Lys‐Thr‐Val‐Arg‐Trp‐ were identical for both bands. These results, and the fact that the two fractions are found in all genetic variants and always have the same relative mobility strongly suggest that the differerences do not lie in the polypeptide chain. Froth the results of hydrolysis by neuraminidase and assay of sialic acid, the major and minor fractions most probably contain two and three sialic acid residues (exclusively N‐acetylneuraminic acid) respectively, thus explaining the different electrophoretic mobilities. The sialic‐acid content has been calculated on the basis of a molecular weight of 77500 as determined both by low‐speed equilibrium ultracentrifugation and by Archibald's method. The following physico‐chemical constants have been established: absorption coefficient at 280 nm, A¹₁^cm_mg/cm³= 1.25 ± 0.01 cm²· mg⁻¹; partial specific volume, v̄= 0.734 cm³· g⁻¹; sedimentation coefficient s°_20,w= 5.15 S; refractiveindex increment, dn/dc = 0.173 cm³· g⁻¹.