Direct spectrophotometric determination of proteins immobilized on bead cellulose and dissolved in cadoxene

Abstract

A direct spectrophotometric method has been developed for the determination of the amount of proteins immobilized on bead cellulose. The method is based on dissolution of conjugates in cadmium tris(ethylenediamine) hydroxide (trivial name, cadoxene) that is optically transparent in the region of the UV spectrum in which proteins absorb. The method developed for the determination of immobilized proteins is simple, rapid, and readily reproducible. The sensitivity of the method is dependent on theA1cm1% value of the free protein and on the amount of bound proteinm (mg protein/g carrier). In the terms ofA1cm1%≥25 andm≥120 mg/g, the sensitivity of the mentioned method was comparable with those obtained by amino acids analyses by the ninhydrin reaction, by the modified Lowry reaction, or by radiometric determination. The possibility of substituting the direct spectrophotometric method for amino acid analysis was statistically evaluated; it does not seem to be rejected at the highest level of significance,P≤1%. The differences in paired observations were significant, but nonetheless proportional over the whole range of protein concentrations, making both methods correlable. The absolute accuracy of the direct spectrophotometric method could be influenced by the change ofA1cm1% value during solvolysis of the conjugate in cadoxene.