Estimation of tryptophyl and tyrosyl exposure in tryptophan-rich proteins by ultraviolet difference spectrophotometry. Lysozyme and Chymotrypsinogen.
Ultraviolet difference absorption spectra produced by ethylene glycol were measured for hen lysozyme [EC 3.2.1.17] and bovine chymotrypsinogen. N-Acetyl-L-tryptophanamide and N-acetyl-L-tyrosinamide were employed as model compounds for tryptophyl and tyrosyl residues, respectively, and their ultraviolet difference spectra were also measured as a function of ethylene glycol concentration. By comparison of the slopes of plots of molar difference extinction coefficients (delta epsilon) versus ethylene glycol concentration for the proteins with those of the model compounds at peak positions (291-293 and 284-287 nm) in the difference spectra, the average number of tyrosyl as well as tryptophyl residues in exposed states could be estimated. The results gave 2.7 tryptophyl and 1.9 tyrosyl residues exposed for lysozyme at pH 2.1 and 2.6 tryptophyl and 3.4 tyrosyl residues exposed for chymotrypsinogen at pH 5.4. The somewhat higher tyrosyl exposure of chymotrypsinogen, compared with the findings from spectrophotometric titration and chemical modification, was not unexpected, because delta epsilon285 was larger than delta epsilon292, and the situation is discussed with reference to preferential interaction of ethylene glycol with the tyrosyl residues and/or side chains in the vicinity of the chromophore in the protein. The procedure employed in the present work seems to be suitable for estimation of the average number of exposed tryptophyl and tyrosyl residues in tryptophan-rich proteins. The effects of ethylene glycol on the circular dichroism spectra of lysozyme at pH 2.1 and chymotrypsinogen at pH 5.4 were also investigated. At high ethylene glycol concentrations, both proteins were found to undergo conformational changes in the direction of more ordered structures, presumably more helical for lysozyme and more beta-structured for chymotrypsinogen.
D000818:Animals; D002417:Cattle; D002645:Chickens; D002919:Chymotrypsinogen; D002942:Circular Dichroism; D005026:Ethylene Glycols; D006863:Hydrogen-Ion Concentration; D009113:Muramidase; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000032:analysis*; D014443:Tyrosine / Q000032:analysis*
D005026:Ethylene Glycols; D014443:Tyrosine; D014364:Tryptophan; D002919:Chymotrypsinogen; D009113:Muramidase