Photochemical protease: site-specific photocleavage of hen egg lysozyme and bovine serum albumin.
Site-specific photocleavage of hen egg lysozyme and bovine serum albumin (BSA) by N-(l-phenylalanine)-4-(1-pyrene)butyramide (Py-Phe) is reported. Py-Phe binds to lysozyme and BSA with binding constants 2.2 +/- 0.3 x 10(5) M-1 and 6.5 +/- 0.4 x 10(7) M-1, respectively. Photocleavage of lysozyme and BSA was achieved with high specificity when a mixture of protein, Py-Phe, and an electron acceptor, cobalt(III) hexammine (CoHA), was irradiated at 344 nm. Quantum yields of photocleavage of lysozyme and BSA were 0.26 and 0.0021, respectively. No protein cleavage was observed in the absence of Py-Phe, CoHA, or light. N-terminal sequencing of the protein fragments indicated a single cleavage site of lysozyme between Trp-108 and Val-109, whereas the cleavage of BSA was found to be between Leu-346 and Arg-347. Laser flash photolysis studies of a mixture of protein, Py-Phe, and CoHA showed a strong transient with absorption centered at approximately 460 nm, corresponding to pyrene cation radical. Quenching of the singlet excited state of Py-Phe by CoHA followed by the reaction of the resulting pyrenyl cation radical with the protein backbone may be responsible for the protein cleavage. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.
Department of Chemistry, University of Connecticut, Storrs, CT 06269-3060, USA. cvkumar@nucleus.chem.uconn.edu
C V
CV
Kumar
A
A
Buranaprapuk
G J
GJ
Opiteck
M B
MB
Moyer
S
S
Jockusch
N J
NJ
Turro
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003035:Cobalt / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D009113:Muramidase / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D010782:Photolysis; D011721:Pyrenes / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism*
D011721:Pyrenes; C032300:hexaamminecobalt(II); D012710:Serum Albumin, Bovine; D003035:Cobalt; D010649:Phenylalanine; C114414:N-(1-phenylalanine)-4-(1-pyrene)butyramide; D009113:Muramidase; D010450:Endopeptidases