Date of Award

1-1-1985

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Abstract

In order to characterize the environmental and ionic nature of the active site of chymotrypsin (Chy), a number of derivatives were prepared and investigated using direct titration, FTIR and fluorescence spectroscopy. Chymotrypsin anthranilate (CA) fluorescence and it quenching was studied as a function of pH, ionic strength and urea concentration. The emission maximum of CA exhibits a blue shift with increasing pH and titrates with a pK(,a) of 7.7. This pK(,a) decreases for CA derivatives with surface carboxyls blocked. The titration of His 57 is likely responsible for this pH shift. Acrylamide was a better quencher at pH 8 than 6, which indicates the active site is more accessible in base. However, quenching by iodide diminished as the pH increased, which suggests that the active site is more negative at high pH. Ionic strength effects on iodide quenching were negative below pH 7 and positive above pH 7 which suggest that the active site changed from cationic to anionic. The variation of CA fluorescence as a function of urea concentration is interpreted as evidence that the active site and the enzyme as a whole exhibits the same stability toward urea. Intensity measurements reveal that the substrate binding pocket remains intact until a concentration of 7M is reached.Effects of solvent environment on the fluorescence of the anthranilate group were studied using ethyl anthranilate (EA), methyl anthranilate (MA) and anthranilic acid (AA). The correlation of fluorescence wavelength of EA with empirical solvent parameters indicates anthranilate fluorescence is a function of the polarity and hydrogen bonding ability of the solvents. The titration of MA and AA showed anthranilate fluorescence is associated with the carbonyl.Direct titrations were carried out on native chymotrypsin and derivatives with 15, 16 and 17 carboxyls selectively blocked. Appropriate plots produced pK(,a)s of 4 and 7, but when the enzymes were denatured with SDS and pK(,a)s shifted to 5.5 and 7. FTIR was employed to titrate the carboxyls in native and modified chymotrypsin. Besides the normal pK(,a)s in the range of 4 to 5, an abnormal pK(,a) appears near 7 in all cases except of the anthranilate and TPCK derivatives. This is evidence for an interaction between Asp 102 and His 57.

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