Preparation and characterization of singly-substituted sulfhydryl derivatives of cytochrome c
Sulfydryl derivatives of horse heart cytochrome c have been prepared by reaction with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3'-dithiopropionate, which modifies lysine epsilon - amino groups. The mixture of products containing cytochromes c derivatized at different lysines was resolved by HPLC using a cation exchange column. The resulting purified mono-derivatives were identified as follows: the sulfhydryl group of the modified lysine was alkylated using (/sup 3/H)-iodoacetic acid, this alkylated derivative was digested with chymotrypsin, and the labeled peptide was isolated by HPLC and its amino acid composition determined. Kinetic analysis of each derivative's reactivity with cytochrome c oxidase revealed a pattern of inhibition similar to that observed for the carboxydinitrophenyl-derivatives of horse heart cytochrome c. By reacting the sulfhydryl group with N-(iodoethyl)trifluoroacetamide, the original charge of the cytochrome c is restored. The effects on the reactivity of cytochrome c with cytochrome c oxidase caused by the change in dipole and by steric hindrance from the derivatization can then be distinguished. These derivatives have the advantage that various labels (photoaffinity, fluorescent, etc.) can readily be attached specifically to each of the modified lysines, and are being used in variety of studies.
- Research Organization:
- Northwestern Univ., Evanston, IL
- OSTI ID:
- 5220757
- Report Number(s):
- CONF-8606151-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ACETAMIDE
ALKYLATION
AMIDES
AMINO ACIDS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARBOXYLIC ACIDS
CARDIOVASCULAR SYSTEM
CHEMICAL COMPOSITION
CHEMICAL REACTIONS
CHROMATOGRAPHY
CYTOCHROME OXIDASE
CYTOCHROMES
ENZYME ACTIVITY
ENZYMES
HAEM DEHYDROGENASES
HEART
HORSES
ION EXCHANGE
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
LYSINE
MAMMALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
OXIDOREDUCTASES
PIGMENTS
PROTEINS
PURIFICATION
REACTION KINETICS
SEPARATION PROCESSES
THIOLS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES