The Denaturation by Urea and Guanidinium Chloride of Trypsin and <i>N</i>‐Acetylated‐Trypsin Derivatives Bound to Sephadex and Agarose

Abstract

Coupling of trypsin to cyanogen‐bromide‐activated Sephadex, but not to agarose, results in a stabilization of the enzyme towards denaturation by urea. Selectively N ‐acetylated trypsin derivatives were prepared and coupled to Sephadex. When a single amino group, which is required for the immobilization reaction, was available for coupling, the insoluble derivative was denatured as the free one. The availability of two or three amino groups for coupling diminished the rate of denaturation considerably. When more than approximately four amino groups were available for coupling, the insoluble derivatives were fully active in 8 M urea. Neither the binding to Sephadex nor that to agarose influenced the denaturation of the enzyme by guanidinium chloride. Differences between the denaturation mechanisms of urea and guanidinium chloride are discussed.