Stabilization of α-chymotrypsin in aqueous organic solvents by chemical modification with organic acid anhydrides

When the primary amino groups of α-chymotrypsin were modified with acetic, propionic, succinic, citraconic and phthalic acid anhydrides, the modifications enhanced the stability of the enzyme in 60% aqueous solutions of 1,4-dioxane, ethanol and acetonitrile. The acetylation of the amino groups resulted in the lowest stabilization, but the citraconylated, propionylated and succinylated forms of α-chymotrypsin exhibited increased stabilities in all the aqueous organic solvents studied. The modification of α-chymotrypsin with phthalic anhydride was accompanied by very extensive activation. The near-UV circular dichroism spectroscopic measurements did not indicate significant structural changes in the acylated forms of α-chymotrypsin, with the exception of the phthalic anhydride-modified enzyme. The spectral changes in the phthalic anhydride-modified enzyme suggest enhanced interactions between the aromatic chromophores and alterations in the disulfide contribution. The improvement in stability may be related to the modifications caused by the reorientation and the increased interactions of the aromatic side-chains, particularly in the case of the phthalic anhydride derivative.