Demonstration of the hydrophilic character of adenylate cyclase following hydrophobic resolution on immobilized alkyl residues. Critical role of alkyl chain length.

The hydrophobic character of the membrane-bound enzyme, adenylate cyclase, was investigated by examining its interaction with a variety of noncharged hydrophobic resins consisting of long chain alkyl groups linked to Sepharose via ether bonds. Either dodecyl or cetyl resins effected the separation of cyclase from more hydrophobic proteins while octyl-, hexyl-, and butyl-Sepharose did not prevent protein aggregation. A critical factor in the resolving capacity of these long chain alkyl resins was their low degree of substitution. Following hydrophobic resolution, gel filtration with Sepharose 6B in the absence of detergent produced a single symmetrical peak of activity in the included volume of the gel duplicating elution patterns with detergent of the unresolved enzyme. Following this step, several fractionation procedures that had previously been ineffective proved successful. Ion exchange chromatography resulted in a single symmetrical peak of activity eluting over a discrete portion of the gradient in contrast to the broad, trailing pattern obtained prior to hydrophobic chromatography. Adenylate cyclase, which previously behaved as a hydrophobic protein, could now be manipulated as a conventional hydrophylic protein. The enzyme now also adhered to a benzyl-Sepharose resin in 1 M NaCl and was eluted with a reverse salt gradient. Resins of similar hydrophobicity such as hexylor butyl-Sepharose, bound the enzyme weakly or not at all, which suggested that II-II interactions played a significant role in cyclase adsorption to the benzyl resin. These results suggest that alkyl residues immobilized on a noncharged matrix behave as solid phase detergents by preventing protein aggregation and simultaneously promoting separation of membrane proteins of varying hydrophobicity. They are likely to provide new and powerful tools in the fractionation and isolation of membrane-bound proteins.