title: Effects of differences in charge and hydrophobicity of surface amino acids of hemoglobins on high-performance gel-permeation chromatography.
abstract: We studied the elution properties of the carboxy and deoxy forms of hemoglobins A, S, and C in gel-permeation high-performance liquid chromatography using TSK-GEL-SW-type columns. Since these hemoglobins have the same molecular mass but different amino acids at the beta 6 position, they are ideal for studies of the effect of charge and hydrophobicity on elution patterns in high-performance gel-permeation chromatography. Although there was a linear relationship between elution volume and logarithm of molecular mass of various proteins, the elution volumes of carboxyhemoglobins were found to be slightly greater than the expected volumes calculated from the molecular mass. The elution volumes of hemoglobins increased in the order of hemoglobins F, A, C, and S in 0.1 M phosphate buffer, pH 7.4, at room temperature. The elution volume of these hemoglobins was also dependent on pH and salt concentration. These results indicate that elution of these hemoglobins was affected by the electrostatic and hydrophobic interactions between hemoglobin molecules and polar sites of silica gel (with silanol groups) of the resin matrix of TSK-G2000-SW. This study may serve as a useful reference for separation and determination of molecular masses of proteins in the native state using gel-permeation liquid chromatography.
authors.affiliation: Children's Hospital, Department of Pediatrics, Philadelphia, PA 19104.
authors.forename: K
authors.initials: K
authors.lastname: Adachi
authors.forename: J Y
authors.initials: JY
authors.lastname: Kim
authors.forename: T
authors.initials: T
authors.lastname: Asakura
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D002263:Carboxyhemoglobin / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D006454:Hemoglobins / Q000032:analysis*; D008970:Molecular Weight
chemical_list: D000596:Amino Acids; D006454:Hemoglobins; D002263:Carboxyhemoglobin