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THE USE OF BENZOPHENONE AS A PHOTOAFFINITY LABEL, LABELING IN p ‐BENZOYLPHENYLACETYL CHYMOTRYPSIN AT UNIT EFFICIENCY
Author(s) -
Campbell Peter,
Gioannini Theresa L.
Publication year - 1979
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1979.tb07787.x
Subject(s) - benzophenone , chemistry , covalent bond , photochemistry , flash photolysis , moiety , titration , photoaffinity labeling , flavin group , hydrogen atom abstraction , photodissociation , derivative (finance) , stereochemistry , enzyme , organic chemistry , binding site , hydrogen , kinetics , reaction rate constant , biochemistry , physics , quantum mechanics , financial economics , economics
— p ‐Benzoylphenylacetyl chymotrypsin, an acyl enzyme derivative containing the benzophenone group in the hydrophobic binding pocket, was prepared and is indefinitely stable at low pH. Photolysis of this covalent derivative leads to loss of enzymic activity and incorporation of the labeling group via formation of a second covalent bond. The efficiency of the photochemical processes is exceptionally high, producing 100% incorporation and at least 92% inactivation. Analysis of active site titration data for the photolyzed enzyme show that at least two different photochemical processes must be involved. Elimination of phosphorescence emission and reduction of UV absorption upon photolysis are consistent with initial hydrogen abstraction by benzophenone triplet state, followed by radical coupling, much as has been observed for the photoreaction of benzophenone with model systems. Photoaffinity labeling of chymotrypsin is also efficiently accomplished using two benzophenone derivatives which bind noncovalently to the enzyme's active site, although the rates of labeling are somewhat less than in the covalent complex.