Raman evidence that the lyoprotectant poly(ethylene glycol) does not restore nativity to the heme active site of horseradish peroxidase suspended in organic solvents.
Resonance Raman spectroscopy was used to interrogate the heme active site of horseradish peroxidase (HRP) lyophilized in the presence and absence of the lyoprotectant poly(ethylene glycol) (PEG; FW 5000; 0-80% w/w) suspended in acetone, chloroform, or acetonitrile. In aqueous solution, Fe(3+)HRP is characterized by a five-coordinate high-spin (5-c HS) heme system. The structure of the heme-active site of HRP in all solvents is perturbed by co-lyophilization of HRP with PEG. Heme active site structural changes are consistent with coordination of water in the distal axial coordination site of the ferric heme iron and disruption of the hydrogen-bond network when the protein is lyophilized in the presence of PEG (>or=60% w/w) in all of the solvent systems studied. Similar active site structural changes were previously observed for HRP in benzene and attributed to a change in the reaction mechanism for HRP in benzene. (Mabrouk, P. A.; Spiro, T. G. J. Am. Chem. Soc. 1998, 120, 10303-10309.) Thus, PEG is proposed to increase the catalytic activity of HRP in nonaqueous media by locking the heme active site into a structure that functions through an alternative catalytic pathway in nonaqueous media.
Guo
Wei
W
Department of Chemistry, Northeastern University, Boston, Massachusetts 02115, USA.
Mabrouk
Patricia Ann
PA
D001665:Binding Sites / Q000187:drug effects; D005612:Freeze Drying; D006418:Heme / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000187:drug effects; D009930:Organic Chemicals; D011092:Polyethylene Glycols / Q000494:pharmacology*; D020011:Protective Agents / Q000494:pharmacology; D020673:Protein Renaturation / Q000187:drug effects*; D012997:Solvents; D013059:Spectrum Analysis, Raman
D009930:Organic Chemicals; D020011:Protective Agents; D012997:Solvents; D011092:Polyethylene Glycols; D006418:Heme; D006735:Horseradish Peroxidase