Fluorescence and optical absorption study of interaction of two water soluble porphyrins with bovine serum albumin. The role of albumin and porphyrin aggregation

Abstract

The interaction of two metal-free water soluble porphyrins (PPh), meso-tetrakis (p-sulfo-natophenyl)porphyrin (TPPS 4) and meso-tetrakis(4-N-methyl-pyridiniumyl)porphyrin (TMPyP), with bovine serum albumin (BSA) was investigated in the pH range from 4.0 to 8.5 using optical absorption and fluorescence spectroscopies. It was found that in this pH range both porphyrins bound to BSA exist in deprotonated free base forms. The binding of PPh quenches the BSA fluorescence. On the contrary, the fluorescence of both monomeric porphyrins increases by the binding. Two types of aggregates were found: those of BSA, and the TPPS 4 aggregates on the surface of the BSA molecule. The TPPS 4 aggregation was observed only when its concentration was higher than that of BSA ( [ TPPS 4] [ BSA] > 1 ), the fluorescence of TPPS 4 being reduced by its aggregation. The TMPyP does not form aggregates. A step-by-step aggregation model was developed to determine the average aggregation numbers of both the BSA (〈 j〉) and the TPPS 4 ( k ̌ 〉 ) from the fluorescence quenching. The (〈 j〉) values vary with pH, BSA concentration and the type of porphyrin from 3 ± 1 to 15 ± 3. The ( k ̌ 〉 ) value is 10 ± 2 at pHs 4.0 and 5.0 and 3 ± 1 at pH 8.5. Binding constants of PPh to BSA ( K h ) are determined as the Stern-Volmer quenching constants of BSA fluorescence. However, the aggregation distorts the binding constant and its real value can be obtained as a limit of the Stern-Volmer one at the lowest possible BSA and PPh concentrations. The K b values depend both on the charge and structure of porphyrin molecules and on the charge and/or the conformation of BSA. The K b values are for TPPS 4 1.5 × 10 8 M −1 at pHs 4.0 and 5.0 and 3.2 × 10 6 M − at pH 8.5 and for TMPyP 7.3 × 10 5 M −1 at pH 5.0 and 1.8 × 10 6 M −1 at pH 8.5.