Photodynamic crosslinking of proteins II. Photocrosslinking of a model protein-ribonuclease A
| Autor: | Pavla Kopečková, Jindřich Kopeček, John D. Spikes, Hui Rong Shen |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Photochemistry
RNase P Biophysics macromolecular substances Bovine pancreatic ribonuclease chemistry.chemical_compound Diethyl Pyrocarbonate Polymer chemistry Animals Organic chemistry Methacrylamide Histidine Radiology Nuclear Medicine and imaging Photosensitizer Ribonuclease Gel electrophoresis Radiation Radiological and Ultrasound Technology biology Chemistry Circular Dichroism Lysine technology industry and agriculture Chemical modification Ribonuclease Pancreatic Cross-Linking Reagents biology.protein Cattle Electrophoresis Polyacrylamide Gel Azide |
| Zdroj: | Journal of Photochemistry and Photobiology B: Biology. 35:213-219 |
| ISSN: | 1011-1344 |
| DOI: | 10.1016/s1011-1344(96)07300-9 |
| Popis: | 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 ϵ-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°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. |
| Databáze: | OpenAIRE |
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