No abstract

Melanins are ubiquitous in nature, yet their functions remain poorly understood, because their structures and properties elude characterization by conventional methods. Since many of the proposed functions of melanins (e.g., antioxidant, pro-oxidant, and radical scavenging) involve an exchange of electrons, we developed an electrochemical reverse engineering methodology to probe the redox properties of melanin. This mediated electrochemical probing (MEP) method (i) characterizes insoluble melanin particles that are localized adjacent to an electrode within a permeable hydrogel film, (ii) employs diffusible mediators to shuttle electrons between the electrode and melanin sample, and (iii) imposes complex sequences of input voltages and analyzes output response characteristics (e.g., currents) to reveal redox properties. Here, we illustrate the versatility of MEP and review results demonstrating that melanins have reversible redox activities, can exchange electrons with various reductants and oxidants, and can quench radicals either by donating or accepting electrons. These results suggest possible biological functionalities for melanin and motivate the use of MEP for characterizing additional (i.e., synthesized) materials whose functions rely on redox properties. More broadly, MEP is revealing a richness to redox activities that has previously been inaccessible to investigation.

Having been activated with glutaraldehyde, modified poly(ethylene terephthalate) grafted acrylamide fiber was used for the immobilization of horseradish peroxidase (HRP). Both the free HRP and the immobilized HRP were characterized by determining the activity profile as a function of pH, temperature, thermal stability, effect of organic solvent and storage stability. The optimum pH values of the enzyme activity were found as 8 and 7 for the free HRP and the immobilized HRP respectively. The temperature profile of the free HRP and the immobilized HRP revealed a similar behaviour, although the immobilized HRP exhibited higher relative activity in the range from 50 to 60 degrees C. The immobilized HRP showed higher storage stability than the free HRP.

Melanins are ubiquitous but their complexity and insolubility has hindered characterization of their structures and functions. We are developing electrochemical reverse engineering methodologies that focus on properties and especially on redox properties. Previous studies have shown that melanins (i) are redox-active and can rapidly and repeatedly exchange electrons with diffusible oxidants and reductants, and (ii) have redox potentials in midregion of the physiological range. These properties suggest the functional activities of melanins will depend on their redox context. The brain has a complex redox context with steep local gradients in O that can promote redox-cycling between melanin and diffusible redox-active chemical species. Here, we performed in vitro reverse engineering studies and report that melanins can redox-cycle with two common redox-active drugs. Experimentally, we used two melanin models: a convenient natural melanin derived from cuttlefish (Sepia melanin) and a synthetic cysteinyldopamine-dopamine core-shell model of neuromelanin. One drug, acetaminophen (APAP), has been used clinically for over a century, and recent studies suggest that low doses of APAP can protect the brain from oxidative-stress-induced toxicity and neurodegeneration, while higher doses can have toxic effects in the brain. The second drug, clozapine (CLZ), is a second generation antipsychotic with polypharmacological activities that remain incompletely understood. These in vitro observations suggest that the redox activities of drugs may be relevant to their modes-of-action, and that melanins may interact with drugs in ways that affect their activities, metabolism, and toxicities.