Beta-thiopropionyl cytochromes c modified at lysyl residues: preparation and characterization of the monosubstituted horse cytochromes c.
beta-Thiopropionyl derivatives of horse cytochrome c singly modified at each of 18 different lysine epsilon-amino groups have been prepared using sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate and purified to homogeneity by high-pressure liquid chromatography. These derivatives were characterized by determination of: (i) the location of the modification; (ii) reduction potentials; (iii) visible and NMR spectra: and by (iv) measurement of electron transfer activity with cytochrome-c oxidase. No significant changes in structure were indicated, except for the ferric forms of the derivatives modified at lysines 72, 73, and 79 which are discussed separately. The electron transfer activity of the beta-thiopropionyl cytochromes c with bovine heart cytochrome-c oxidase was decreased to extents dependent on the position of the modification. Aminoethylation, a secondary modification which reverses the charge change, restored the electron transfer rate to that observed with the unmodified cytochrome c, irrespective of the location of the primary modification. These results afford a direct experimental demonstration that alterations in kinetics with physiological electron transfer partners resulting from modifications which cause a change of the charge of surface side chains are solely due to the electrostatic effects. Of the many chemically modified cytochromes c prepared to date, the singly substituted beta-thiopropionyl cytochromes c are likely to be particularly useful as the thiol allows covalent linkage of any sulfhydryl-reactive reagent to a well-defined location on the protein surface by a simple procedure, even when the secondary modifier is relatively unstable, a crucial advantage not otherwise readily achieved.
Theodorakis
J L
JL
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.
Armes
L G
LG
Margoliash
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001643:Bridged Bicyclo Compounds; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000138:chemical synthesis* / Q000737:chemistry* / Q000302:isolation & purification; D003576:Electron Transport Complex IV / Q000737:chemistry; D006736:Horses; D008239:Lysine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D013056:Spectrophotometry, Ultraviolet
D001643:Bridged Bicyclo Compounds; D003574:Cytochrome c Group; D008715:Methionine; D003576:Electron Transport Complex IV; D008239:Lysine; C028381:monobromobimane