Abstract
Purpose :
Tyrosinase (Tyr) initiates the melanogenesis pathway by producing dopachrome (DC). The isolation of DC is essential to having a quantitative characterization of Tyr function with regard to mutations linked to oculocutaneous albinism 1 (OCA1). Previously we offered a method of DC isolation to allow current research to obtain this quantitative analysis by immobilizing Tyr to magnetic beads (Tyr-MB). Here we show increased thermostability of Tyr bound to Ni-NTA magnetic beads (MB) and have characterized and performed experiments on Tyr-MB to further our understanding of the role of wildtype and OCA1-related mutant Tyr within the melanogenesis pathway.
Methods :
Whole T. ni larvae expressing recombinant truncated human Tyr (residues 19-469) was purified using Immobilized Metal Affinity and Size Exclusion Chromatography. 100 µL of MB were incubated with Tyr for 30 minutes at room temperature to allow Tyr to bind. L-DOPA substrate (12, 6, 3, 1.5, 0.75, 0.38, 0.19, or 0.09 mM) was then incubated (1:1) with Tyr-MB complexes for 30 min. at different temperatures (25, 31, 37, 43, or 50 oC) to produce DC. After isolating DC with a magnetic separator, Tyr activity for DC formation was tracked by colorimetric activity (475 nm). Bare MB and Tyr-MB were imaged and analyzed using Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM).
Results :
A dose-dependent hill plot (R2 = 0.99) using different L-DOPA concentrations confirmed similar behavior between Tyr-MB and native Tyr (positive cooperativity; hill coefficient: 1.65 and 1.71 respectively; L-DOPA concentration that produces 50% of maximal DC production: 3.18 and 3.01 mM respectively.) A temperature dependence curve revealed a direct correlation (R2 = 0.97) of Tyr-MB DC production with temperature. Under 50 oC, Tyr-MB remained functional and produced significantly more orange-brown DC than native Tyr (clear solution) as the MB increased Tyr thermostability. AFM and TEM showed the “texture and roughness” of bare MB and Tyr-MB and the relative diameter of Tyr-MB (168.2 ± 24.4 nm).
Conclusions :
The analysis suggests using MB for DC isolation to obtain a quantitative characterization of Tyr function is an efficient approach as it increases the thermostability of Tyr. This approach will further illuminate the role of OCA1 mutant Tyr with regard to melanogenesis and open doors for treating albinism-related disorders.
This is a 2021 ARVO Annual Meeting abstract.