Total synthesis of horse heart apocytochrome c by conformation-assisted condensation of two chemically synthesized fragments.
A fully synthetic peptide, corresponding to the entire 104-residue sequence of horse heart apocytochrome c with Met65 replaced by homoserine, has been obtained by an original conformation-assisted three-fragment condensation procedure. The method involves the selective joining of two synthetic fragments, namely residues 1-65 of the apopeptide with Met65 replaced by homoserine lactone and residues 66-104 of the protein in the presence of fragment 1-25 of the native heme-containing peptide. The joining conditions have been optimized with regard to solvent, pH and possible influence of additives. The presence of radical scavengers and the complete exclusion of oxygen were found essential in order to prevent oxidative side reactions. A sensitive method based on reverse-phase HPLC has been used to monitor the course of the reaction. Condensation yields up to 80% were obtained. The data obtained by this new three-fragment rejoining approach are discussed and compared to those of a similar two-fragment condensation procedure. Our data demonstrate how the folding properties of large synthetic peptide fragments, organized in a complex, can be utilized to extend the presently improved solid-phase peptide methods to the synthesis of a functioning protein with more than 100 residues.
Vita
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Institute of Industrial Chemistry, University of Padua, Italy.
Gozzini
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Di Bello
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D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000096:biosynthesis* / Q000235:genetics; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics; D045304:Cytochromes c; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D001059:Apoproteins; D003574:Cytochrome c Group; D045304:Cytochromes c