Pegylated catalase as a potential alternative to treat vitiligo and UV induced skin damage

Abstract

The metabolic function of catalase (CAT) is to prevent oxidative damage to tissues through the hydrolysis of hydrogen peroxide, which is a strong oxidizing agent. It has been suggested as an alternative to treat skin diseases related to oxidative stress, such as vitiligo. Owing to the instability associated to the protein nature, topical use of CAT is challenging and, in this sense, PEGylation can be an interesting alternative. Here, we conjugated CAT to methoxy-poly(ethylene oxide) (mPEG) of 10, 20 and 40 kDa, by means of a nucleophilic attack of ε-amino groups to an electron-deficient carbonyl group of the reactive PEG, resulting in site specifically PEGylated bioconjugates. PEGylation yields ranged from 31% ± 2% for CAT-PEG40 to 59% ± 4% for CAT-PEG20 and were strongly affected by the reaction pH owing to the protonation/deprotonation state of primary amines of lysine and N-terminal residues. PEGylated conjugates were purified by size-exclusion chromatography (purity > 95%) and characterized by circular dichroism. Irrespectively of MW, PEG did not affected CAT secondary and tertiary structure, but a decrease in specific activity was observed, more pronounced when PEGs of higher MWs were used. However, this loss of activity is compensated by the increased long-term stability, with a gain of >5 times in t_1/2. In vitro antioxidant activity of CAT-PEG20 showed complete elimination of lipid peroxidation at the skin upper layer (stratum corneum) suitable for a topical use to treat vitiligo, as well as other skin conditions related to oxidative stress.

Keywords: Antioxidant activity; Bioconjugation; Catalase; PEGylation; UV induced skin damage.