Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability

Author(s)

Santos, Joao H. P. M. ; Feitosa, Valker A. ; Meneguetti, Giovanna P. ; Carretero, Gustavo ; Coutinho, Joao A. P. ; Ventura, Sonia P. M. ; Rangel-Yagui, Carlota O.

• Document type: Journal article
• Source: BIOSENSORS-BASEL; v. 12, n. 2, p. 13-pg., 2022-02-01.

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 degrees C and 25 degrees C. (AU)

• FAPESP's process: 16/22065-5 - N-terminal pegylation of proteins and purification by aqueous two-phase systems
• Grantee: Carlota de Oliveira Rangel Yagui
• Support Opportunities: Regular Research Grants
• FAPESP's process: 18/25994-2 - Development of novel platforms for PEGylation of proteins with therapeutic potential using microfluidics
• Grantee: João Henrique Picado Madalena Santos
• Support Opportunities: Scholarships in Brazil - Post-Doctoral