Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability

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.

Keywords: bioconjugation; cytochrome-c; long-term stability; lysine PEGylation.

Figure 1

Representation of PEGylation reaction of cytochrome c (Cyt-c) for the attachment of 4 and 8 PEG molecules. (a) Schematic overview of PEGylation reaction in primary amine groups of Cyt-c with methoxy polyethylene glycol N-hydroxysuccinimide (mPEG-NHS). Graphical representation of theoretical lysine residues (Lys) on Cyt-c (PDB code 1HRC) and its (b) 4-PEGylated, and (c) 8-PEGylated counterparts using the PyMOL^® software. Gray lines in the Cyt-c PEGylated forms represent attached mPEG chains.

Figure 2

Effect of pH on cytochrome c (Cyt-c) PEGylation yield (%) at different pH values: (a) pH = 7, (b) pH = 8, (c) pH = 9, (d) pH = 10, (e) pH = 11 and (f) pH = 12. The PEGylation reaction was performed in 0.1 M potassium phosphate buffer, 1:25 molar proportion (Cyt-c:mPEG-NHS, 5 kDa), during 30 min: n.d., not detected.

Figure 3

(a) Effect of protein:mPEG-NHS molar ratio on cytochrome c (Cyt-c) PEGylation yield (%). The PEGylation reaction was performed at pH 7 for 30 min. (b) Effect of reaction time on Cyt-c PEGylation yield. The PEGylation reaction was performed in 0.1 M potassium phosphate buffer (pH 7), 1:25 molar proportion (protein:mPEG-NHS): n.d., not detected.

Figure 4

Chromatogram (a) and electrophoretic profile (b) of cytochrome c (Cyt-c) after PEGylation reaction under best experimental conditions: pH 7, 1:25 molar ratio (Cyt-c:mPEG-NHS) and reaction time 30 min: Cyt-c-PEG-4, 4 mPEG molecules attached; Cyt-c-PEG-8, 8 mPEG molecules attached.

Figure 5

Experimental far-UV CD spectra (black lines) of Cyt-c (a), Cyt-c-PEG-4 (b), and Cyt-c-PEG-8 (c) in 0.01 M sodium phosphate buffer (0.14 M NaCl, pH 7.4); sample concentration ranged from 6 to 15 µM [34]. Theoretical CD spectra (red lines) of native and PEGylated Cyt-c conjugates calculated from experimental spectra using BestSel algorithm, and fit residuals.

Figure 6

Stability of native and PEGylated forms of cytochrome c (Cyt-c) stored at 4 °C (a) and 25 °C (b). Residual peroxidative-like activity of Cyt-c (control) was determined by the catalytic oxidation of 50 µM ABTS in the presence of 0.5 mM hydrogen peroxide. The concentration of native and PEGylated Cyt-c forms was 10 µM in 0.01 M phosphate buffer (0.14 M NaCl, pH 7.4).