Difference Spectrum of Diisopropylphosphoryltrypsin versus Trypsin

Abstract

DIFFERENCE spectra of diisopropylphosphoryl-α-chymotrypsin versus α-chymotrypsin (Fi 1) and of monoacetyl-α-chymotrypsin versus α-chymotrypsin have been described in earlier papers^1–3. Evidence was presented^3,4 which indicates that these difference spectra, with maxima at 290 mµ, are due to conformational changes brought about by interaction of the substrate with chymotrypsin. In this communication, the difference spectrum of diisopropylphosphoryl-trypsin (DIP-trypsin) versus trypsin will be described. A solution containing approximately 1 mg/ml. of trypsin (twice crystallized, salt-free, Worthington Biochemical Corporation, Freehold, New Jersey) was adjusted to pH 7.4. Exact aliquots of this solution were pipetted into matched cells which were placed into the reference and sample compartment of a Cary model 14 spectrophotometer. A 20-fold excess of diisopropylphosphorofluoridate (DFP) in anhydrous isopropanol was added to the sample cell and an appropriate amount of isopropanol to the reference cell. The resulting difference spectrum, with a major peak at 287 mµ, can be seen in Fig. 1. Spectra were recorded 4–20 min after the addition of DFP. No further spectral changes occur after 4 min, indicating that the observations are not due to changes in the control solution, such as autolysis of trypsin.