Comparative structural and functional studies of Sturgeon Hemoglobin (Acipenser persicus and Acipenser stellatus)

Abstract

Hemoglobin (Hb) variability is a commonly used index of phylogenetic differentiation and molecular adaptation in fish enabling them to adapt to different ecological conditions. In this study, the characteristics of Hbs from two Sturgeon species of the Southern Caspian Sea Basin were investigated. After extraction and separation of hemoglobin from whole blood, the polyacrylamide gel electrophoresis (SDS-PAGE), cellulose acetate electrophoresis, and isoelectric focusing (IEF) were used to confirm Hb variabilities in these fishes. We showed that although both species have variable Hbs with different isoelectric points, their dominant Hbs can be identified. Ion exchange on CM-cellulose chromatography was used for purification of the dominant Hbs from these fishes. The accuracy of the methods was confirmed by IEF and SDSPAGE. Spectral studies using fluorescence spectrophotometery indicated that although the Hbs from these fishes had similar properties. A comparative study of Hbs alpha-helix secondary substructures was performed by circular dichroism spectropolarimetry (CD) analysis. The thermal denaturation properties of the Hbs were investigated by differential scanning calorimetry (DSC) .UV–vis spectrophotometery was also utilized to measure oxygen affinity of Hbs by sodium dithionite. Oxygen affinities of these Hbs were compared using Hb–oxygen dissociation curves. Together, these results demonstrate a significant relationship between oxygen affinity, hydrophobicity, themostability and alpha-helix secondary substructures of fish hemoglobins and environmental partial pressure of oxygen.