The determination of molecular weight of proteins by gel permeation chromatography in organic solvents.
Very amphiphilic proteins with a high tendency to self-aggregate may exist in an aggregated form even in the presence of detergents or denaturants. In order to mitigate the tendency towards self-association, it is necessary to eliminate the amphiphilicity of these proteins. In order to achieve this end, proteins were modified covalently, first by dinitrophenylation followed by permethylation, which rendered the proteins tested soluble in nonpolar organic solvents, such as chloroform/methanol (4/1, v/v). The permethylated 2,4-dinitrophenyl-proteins were then chromatographed using Sepharose CL-6B in chloroform/methanol (4/1, v/v). For ten commonly used molecular weight marker proteins, a single symmetrical peak was obtained in the elution profile of the modified proteins, indicating that these products are monodisperse with respect to molecular size. The one exception was gamma globulin which has two polypeptide chains, and thus the elution profile showed two symmetrical peaks. The KD value was found to be a linear function of the logarithm of the molecular weight of the parent protein. From the calibration line generated from the molecular weight marker proteins, the technique was applied to five highly amphiphilic proteins: bacteriorrhodopsin, uricase, insecticyanin, apolipoprotein B of plasma low density lipoproteins, and band 3 of human erythrocyte membrane, for which the following apparent molecular weights were obtained: 25,000, 31,000, 23,400, 19,000, and 85,000, respectively. These values match the molecular weights obtained from the amino acid sequence in those cases in which the sequence is known. Thus, permethylation in conjunction with organic media disrupts the major driving forces for tertiary and quarternary structure formation in aqueous media, namely, the hydrophobic effect, salt bridges, and hydrogen bonding with the solvent.
D019297:2,4-Dinitrophenol; D002850:Chromatography, Gel / Q000379:methods; D004140:Dinitrophenols; D008745:Methylation; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis*; D012997:Solvents
D004140:Dinitrophenols; D011506:Proteins; D012997:Solvents; D019297:2,4-Dinitrophenol