Photodynamic crosslinking of proteins. II. Photocrosslinking of a model protein-ribonuclease A.
Illumination of bovine pancreatic ribonuclease A (RNase A) in solution in the presence of rose bengal as a photosensitizer resulted in the progressive formation of enzyme dimers, trimers, tetramers and higher oligomers, as measured by gel electrophoresis and size exclusion chromatography. Oxygen was necessary for crosslink formation, and azide inhibition studies indicated that singlet oxygen was involved in the process. Chemical modification of His residues (with diethyl pyrocarbonate) and/or Lys residues (with acetic acid N-hydroxysuccinimide ester) in the enzyme decreased crosslinking, suggesting the participation of these two amino acid residues in the reaction. Met and cystine residues did not appear to be involved. Similar studies have shown that model N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing epsilon-aminocaproic acid side chains terminating in His or Lys residues are photodynamically crosslinked via His-His or His-Lys interactions. Treatment of crosslinked RNase A and its His, Lys and Lys-His derivatives for 5 min at 97 degrees C in a dithiothreitol-sodium dodecyl sulfate mixture efficiently ruptured a major part of the photodynamically formed crosslinks; treatment with the detergent alone had no effect. Similar results were obtained with the crosslinked amino acid-containing HPMA copolymers, suggesting that photodynamic crosslinks involving His-His and His-Lys interaction are chemically the same in RNase A and the copolymer model.
Shen
H R
HR
Department of Bioengineering, University of Utah, Salt Lake City 84112, USA.
Spikes
J D
JD
Kopecková
P
P
Kopecek
J
J
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004047:Diethyl Pyrocarbonate; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine; D008239:Lysine; D010777:Photochemistry; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*
D003432:Cross-Linking Reagents; D006639:Histidine; D012259:Ribonuclease, Pancreatic; D008239:Lysine; D004047:Diethyl Pyrocarbonate