Abstract
Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30–40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C.
Highlights
Cytochrome c (Cyt-c) is a small protein with 104 amino acids and a molecular weight of about 12 kDa
Regarding the design of site-specific polymer-protein conjugates, the selection of appropriated conditions for the PEGylation reaction is the first step towards obtaining a successful site-specific PEGylated product
Terhoexcidoen.cTenhteractoinocneonftrnaatitoivneoafnndative PEGaynladtePdEGCytla-ctefdorCmyst-wc afosr1m0sμwMasin10.μ01MMinp0h.o01spMhaptehobsupffhearte(0b.1u4ffMer N(0a.1C4l,MpHNa7C.4l),.pH 7.4). These results demonstrate that site-specific PEGylation is a valuable strategy to increase cytochrome c (Cyt-c) stability and shelf life
Summary
Cytochrome c (Cyt-c) is a small protein with 104 amino acids and a molecular weight of about 12 kDa. Cytochrome c (Cyt-c) is a small protein with 104 amino acids and a molecular weight of about 12 kDa It is a heme protein involved in mitochondrial electron transfer, even though it is not considered a natural enzyme; it catalyzes several chemical reactions, including hydrogen peroxide reduction, aromatic oxidation, hydroxylation, epoxidation and N-demethylation [1]. Based on the broad heterogeneity obtained in biotransformation reactions catalyzed by Cyt-c, along with its high reactivity towards different substrates and its electron transfer capability, this protein has been recently explored as a biosensor of hydrogen peroxide, nitric oxide and polycyclic aromatic hydrocarbons [1,2,3]. Its instability results from the fragile structure shared by most proteins
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