Fibronectin (FN) is an essential protein of the extracellular matrix (ECM) needed in wound healing. In chronic wounds, the high levels of... Show moreFibronectin (FN) is an essential protein of the extracellular matrix (ECM) needed in wound healing. In chronic wounds, the high levels of protease in the wound bed lead to excessive degradation of fibronectin, which delays the healing process. Developing a proteolytically stable and functionally active form of FN is the main purpose of this research. Conjugating of proteins to polyethylene glycol (PEG) or PEGylating proteins showed more proteolytic stability than native FN degradation without perturbing their activity. The goal of this study was to compare the proteolysis of native and PEGylated fibronectin with different PEG length. Fibronectin was purified from human blood plasma and conjugated to PEG Diacrylate (PEGDA) and other types of PEG to yield the PEGylated human plasma fibronectin (PEG-HPFN). α-chymotrypsin and neutrophil elastase were used as digestion enzyme during degradation reaction. The proteolysis reaction was stopped at different time points with protein inhibitor phenylmethanesulfonylfluoride (PMSF). The samples were analyzed by SDS-PAGE followed by silver staining or immunblotting with antibodies specific to human fibronectin. Densitometric analyses of the polyacrylamide gels or the blots demonstrated that PEG-HPFN was more stable than native HPFN. The results demonstrate that PEGylation is a robust approach for stabilizing fibronectin. Future studies into activity of PEGylated proteins as well as the role of PEGylation factors such as extent of PEGylation or PEG length on activity will provide novel strategies of mitigating fibronectin degradation in chronic wounds. M.S. in Chemical Engineering, July 2013 Show less
