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The Thiopropionyl Derivatives of Cytochrome C

  • Janice L. Theodorakis
  • Lyman G. Armes
  • E. Margoliash

Summary

ß-Thiopropionyl derivatives of horse cytochrome c singly-modified at each of 19 different lysine e-amino groups have been prepared by a procedure involving the intermediate formation of a biotinylated compound which allows the bulk separation of the modified from the unmodified protein by binding to avidin-biotin-Agarose. Subsequent purification of individual mono-derivatives was achieved using high pressure liquid chromatography. The lysine modified on each mono- derivative was determined following reaction of the sulfhydryl with [3H] —iodoacetate, by digestion with Chymotrypsin, separation of the resulting peptides by HPLC, isolation of the radioactive peptide and its identification by amino acid composition analysis. Polarographic assays of the reaction of the ß-thiopropionyl derivatives with the oxidase of beef heart mitochondrial particle preparations showed that, in the high affinity phase of the system, the order of decreasing activities was qualitatively similar to that observed earlier for other chemical modifications changing the charge of these lysines from positive to negative, specifically the carboxydinitrophenyl-derivatives [Brautigan et al.(1978) J. Biol. Chem. 253, 130-159] of horse cytochrome c. These ß-thiopropionyl derivatives have the advantage over previous sets of derivatives of being very reactive with a variety of reagents such that an already purified mono-substituted derivative of cytochrome c can be further modified to yield a well characterized singly-substituted derivative of cytochrome c,with the second modifier at a known location, for use in fIuorescence, EPR, and other studies. The major limitation encountered is the instability of the ß-thiopropionyl derivatives. However, this can be overcome by careful storage conditions and by reacting the derivatives with the secondary modifier as soon as the desired derivative is purified to homogeneity.

Keywords

High Pressure Liquid Chromatography Secondary Modifier Cation Exchange Column Photoaffinity Label Amino Acid Composition Analysis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • Janice L. Theodorakis
    • 1
  • Lyman G. Armes
    • 1
  • E. Margoliash
    • 1
  1. 1.Department of Biochemistry, Molecular Biology and Cell BiologyNorthwestern UniversityEvanstonUSA

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