Interaction of chlorin p6 with bovine serum albumin and photodynamic oxidation of protein.
The binding of chlorin p6, a photosensitizer having basic tetrapyrrole structure, to bovine serum albumin (BSA) and oxidation of the protein following photodynamic treatment is studied. The Stern-Volmer plot indicates that binding of chlorin p6 to BSA was of single class. Binding parameters, binding association constant and number of binding sites, were found to be 1.62+/-0.27 x 10(5)M(-1) and 1.086+/-.019, respectively. Photodynamic oxidation of protein was studied by (i) loss of intrinsic fluorescence of protein, (ii) protein carbonyl formation, (iii) protein hydroperoxide (iv) formation of TCA soluble amino groups and (v) SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Intrinsic protein fluorescence was observed to decrease almost linearly as a function of irradiation time at a fixed concentration of chlorin p6 and with increasing concentration of chlorin p6 at fixed time of irradiation. Protein carbonyl and hydroperoxide formation was found to increase with increasing photodynamic treatment. No significant increase in 5% TCA soluble amino groups was observed. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) reveals that photodynamic treatment of BSA in presence of chlorin p6, rose bengal and riboflavin causes non-specific fragmentation of protein. Photodynamic carbonyl formation by chlorin p6 was not inhibited by sodium formate (100 mM) or mannitol (25 mM) but was significantly inhibited by sodium azide (2 mM). Protein carbonyl formation increased almost 90% when H2O was replaced by D2O. The results show that chlorin p6 induced photodynamic oxidation of BSA was mainly mediated by singlet oxygen.
Bose
Biplab
B
Biomedical application section, Block-D, Center for advanced Technology, Indore 452013, India. bbose@cat.ernet.in
Dube
Alok
A
D017666:Deuterium Oxide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D005561:Formates / Q000494:pharmacology; D008027:Light; D008353:Mannitol / Q000494:pharmacology; D010084:Oxidation-Reduction; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011166:Porphyrins / Q000737:chemistry*; D012256:Riboflavin / Q000737:chemistry; D012395:Rose Bengal / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D019810:Sodium Azide / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014238:Trichloroacetic Acid / Q000737:chemistry
D005561:Formates; D017319:Photosensitizing Agents; D011166:Porphyrins; C030544:formic acid; D012395:Rose Bengal; C072905:chlorin p6; D012710:Serum Albumin, Bovine; D008353:Mannitol; D014238:Trichloroacetic Acid; D019810:Sodium Azide; D017666:Deuterium Oxide; D012256:Riboflavin