General aspects of hydrophobic chromatography. Adsorption and elution characteristics of some skeletal muscle enzymes.
If the degree of substitution of Sepharose 4 B with alpha-alkylamines is varied gels of different hydrophobicity are produced. Proteins can be adsorbed when a critical hydrophobicity (ca. 10-12 alkyl residues/Sepharose sphere) is reached. The enzymes phosphorylase kinase, phosphorylase phosphatase, 3',5'-cAMP dependent protein kinase, glycogen synthetase, and phosphorylase b are successively adsorbed as the hydrophobicity of the Sepharose is increased. The capacity of the gels for these enzymes and protein in general increases exponentially reaches plateau values as a function of the degree of substitution. There is no indication of a restriction of the hydrophobic centers for a given protein. The critical hydrophobicity needed to adsorb proteins can either be otained in the above manner or by elongation of the employed alkylamine at a constant degree of substitution. Additonally, as the hydrophobicity of a gel is increased higher binding forces result and desorption of proteins requires an augmentation of the salt concentration in the elution buffer. Elution of proteins from a hydrophobic matrix can be described in terms of salting-in phenomena since desorption is dependent on the type of salt employed and not on the ionic strength alone. This also rules out ionic interactions as a major factor in adsorption per se. By rationally controlling the hydrophobicity of a Sepharose gel the adsorption and elution of a protein may be thus establised that its purification or elimination can be optimally performed.
D000588:Amines; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel / Q000379:methods*; D006006:Glycogen Synthase / Q000302:isolation & purification; D009124:Muscle Proteins / Q000302:isolation & purification*; D009132:Muscles / Q000201:enzymology*; D009994:Osmolar Concentration; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification; D010764:Phosphorylase Kinase / Q000302:isolation & purification; D006005:Phosphorylases / Q000302:isolation & purification; D011494:Protein Kinases / Q000302:isolation & purification; D012685:Sepharose
D000588:Amines; D009124:Muscle Proteins; D012685:Sepharose; D006005:Phosphorylases; D006006:Glycogen Synthase; D011494:Protein Kinases; D010764:Phosphorylase Kinase; D010744:Phosphoric Monoester Hydrolases