Abstract
Purpose :
Human tyrosinase (Tyr), comprised of a binuclear copper active site, is one of three Type 1 membrane-associated glycoenzymes found in human melanocytes involved in melanin production. Tyr mutations have been linked to oculocutaneous albinism 1 (OCA1) and other genetic diseases. Tyr initially catalyzes L-DOPA to form DOPAchrome initiating the melanogenesis pathway. Current research is harmed by the absence of quantitative characterization of DOPAchrome-related products to understand the role of genetic mutations in Tyr function. Here we isolate and characterize DOPAchrome-related products from the reaction catalyzed by purified Tyr.
Methods :
Whole Trichoplusia ni (T. ni) larvae were utilized for expressing the recombinant truncated Tyr (residues 19-469). To achieve purification, Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC) were performed. Tyr was bound to increasing volumes (50, 100, 150, 200 µL) of Ni-NTA magnetic beads (MB) by incubating the Tyr sample (2.2 mg/mL) with MB at room temperature for 30 minutes and the supernatant was removed using a magnetic separator. Subsequently, Tyr activity for DOPAchrome formation was tracked by colorimetric activity (475 nm) at 37 oC for 30 minutes after adding the L-DOPA substrate. Dynamic Light Scattering (DLS) was used to measure the hydrodynamic diameters of Tyr and DOPAchrome derived products. MB and Tyr-bound MB were analyzed using Atomic Force Microscopy (AFM), using a poly-L-lysine coated substrate to fix the negatively charged hydrophilic layer of the particles.
Results :
Tyr binding is directly correlated (R2 = 0.78) to volume of MB at a Tyr binding rate of 0.0024 µg/µL2. Colorimetric activity showed direct correlation (R2 = 0.98) of DOPAchrome production (0.114, 0.159, 0.192, 0.216 o.u.) as the volume of Tyr-bound MB increased respectively with a rate of 0.0007 o.u./µL. DLS revealed the hydrodynamic diameters for Tyr (8.04 nm) and DOPAchrome products (0.87, 63.0, and 1200.0 nm). AFM images revealed surface “texture and roughness” of the bound/unbound MB.
Conclusions :
The analysis suggests this is an efficient approach to achieve DOPAchrome formation and isolation. In light of quantitative melanin in vitro characterization, it will further illuminate the activity of OCA1 mutant variants in their function within the melanogenesis pathway and open doors to the treatment of albinism-related genetic disorders.
This is a 2020 ARVO Annual Meeting abstract.