Equilíbrio, análise conformacional e atividade biológica de hemoglobina complexada a dopamina

Abstract

Hemoglobin is the main protein in the transport of O2 in vertebrates. In recent years studies have reported its expression in atypical sites such as dopamine-producing cells, a neurotransmitter responsible for several functions, including regulation of movement, in the nigra substance of the brain, its selective degeneration leads to Parkinson's disease, being the second most common neurodegenerative disorder that affects 2 to 3% of the world population over 65 years of age and 3.3% in Brazil. In this sense this project aimed to evaluate the interaction of dopamine with hemoglobin in vitro and its plausible effect on the biological response the respiratory pigment. Tests were conducted to understand and characterize thermodynamically and conformationally the interaction of Hb-DA by uv-vis spectrophotometry, spectrofluorimetry and cyclic voltammetry. The complex formation was confirmed by the three techniques, revealing an average value for Kd of 275 Mol.L-1. Analysis by Benesi-Hildebrand, Scatchard, Maitra and Hill confirmed the formation of the Hemoglobin-Dopamine complex in a 1:1 ratio, also discarding homocooperativity or heterogeneity for the 2 binding sites found for each protein monomer. The other thermodynamic parameters obtained by fluorimetry showed values of G° = -15.7 kJ mol-1 at 25 °C, H° +8.8 kJ mol-1 andS° +82.2 J mol-1 K-1. This suggests a entropically-directed and spontaneous complex formation, which was reinforced by the low contribution of ionic interactions and saline bridges in assays with pH and NaCl changes. Testing the complex formation with ANS as a hydrophobic probe, no mean conformational change could be identified, although a negative influence of DA in Hb Tm seems to be happened. Complimentarily, DA had a negative effect on the peroxidase activity of Hb against H2O2 as substrate, thus suggesting that this interaction may have an impact on the cellular mechanisms of oxidative stress in dopaminergic neurons that express Hb in brain.