Abstract:
Melanin is a very widely studied biopolymer, which gives color to our eyes, skin, hair etc and whose complex structural hierarchy has puzzled researchers over the past century. The biogenesis pathway of melanin had been investigated previously and found that the enzyme tyrosinase has a crucial role in the oxidation of precursors (tyrosine and 3,4-dihydroxyphenylalanine) that facilitates further oxidation to the indolic species of dihydroxyindole (DHI) and dihydroxyindole-2-carboxylic acid (DHICA), which take part in polymerization. Though a general mechanism for consecutively produced reactants in this polymerization pathway was proposed back in 1926, kinetics is not entirely known yet. The current project aims to investigate the kinetics of melanin polymerization from the precursor molecule, 3,4-dihydroxyphenylalanine (DOPA), in the presence and absence of tyrosinase using spectroscopic methods. Due to the broad and interfering absorption spectra of melanin and intermediates, the obtained kinetics of several intermediates from absorbance data wrongly estimates the original value. We have measured kinetics using resonance Raman (RR) spectroscopy which probes molecular vibrational energy levels and thus is intrinsically of a higher spectral resolution than that of absorption spectroscopy. In the current study a novel reduced reaction model arises from RR derived kinetics which sheds considerable amount of light on the current understanding of melanin synthesis pathway. The rate constants obtained by using the above mentioned models of the kinetics of various intermediates and products, with and without the enzyme, are discussed with their significance. A major finding in this work is the evidence for the regulatory action of the enzyme tyrosinase on the heterogeneity of the polymers formed. The latter part of the project looks at the vibrational spectroscopy of various cell types with and without melanin.
