Changes in conformation and immunological activity of ovalbumin during its modification with different acid anhydrides.

In order to probe the cause and nature of conformational changes induced by the chemical modification of amino groups in proteins, five acylated derivatives of ovalbumin namely 21% acetylated, 32% succinylated, 90% butyrated 92% succinylated, and 95% acetylated ovalbumins were prepared and their molecular and immunological properties were systematically investigated. As evidenced by the ultraviolet difference spectral, solvent perturbation, gel filtration, and viscosity data, acylation of the amino groups produced a definite conformational change in native ovalbumin whose extent was higher for higher degrees of chemical modification. The solvent pertubation data showed an exposure of 0.5 tryptophan and 3 tyrosine residues in native ovalbumin; the exposure increased to 1 tryptophan and about 5 tyrosine residues in the maximally modified proteins (i.e. 90% butyrated, 92% succinylated, and 95% acetylated ovalbumins). The Stokes radius (2.7 nm) and intrinsic viscosity (3.9 ml/g) of ovalbumin increased, respectively, to about 3.4 nm and 7.7 ml/g upon acylation of its 18 lysine residues; the intrinsic viscosity of 95% acetylated ovalbumin was 7.2 ml/g. The reduced viscosity of ovalbumin (4.2 ml/g) which remained unaltered on raising the pH to pH 11.2, increased to 7.9 ml/g on succinylation of 18 lysine residues. On raising the ionic strength from 0.15 to 1.0, the value decreased from 7.9 to 6.2 ml/g. These observations taken together with the fact that the intrinsic viscosities of 92% succinylated and 90% butyrated ovalbumins are identical, argue against the presently prevalent proposal that electrostatic effects alone are responsible for the disruption of native protein conformation during chemical modification. The immunological activity of ovalbumin towards rabbit anti-ovalbumin expectedly decreased with acylation of its amino groups but the three maximally modified ovalbumins retained 40% immunological activity. This taken along with the spectral and viscosity data showed substantial native structure (format) in the three maximally acylated derivatives. The rabbit antiserum against 95% acetylated ovalbumin did not cross-react with acetylated lysozyme and reacted poorly with the native and 92% succinylated ovalbumins suggesting that the antigenic make-up of the three maximally modified ovalbumins is different.