Effects of dithiothreitol on protein activity unrelated to thiol-disulfide exchange: for consideration in the analysis of protein function with Cleland's reagent

Anal Biochem. 2000 Jun 15;282(1):102-6. doi: 10.1006/abio.2000.4557.

Abstract

Dithiothreitol (DTT) is widely used to reduce disulfide bonds in the analysis of protein structure and function. However, thiol-disulfide exchange is not the only mechanism whereby DTT can alter protein function. We observe that DTT diminishes the carbohydrate binding activity of a cysteineless mutant of pigpen as well as it inhibits the intact molecule. Lack of inhibition by threitol, a derivative of the four-carbon sugar threose, indicates that the thiol groups of DTT are required for inhibition, and also that DTT is not acting as a simple carbohydrate competitor. Moreover, inhibition of pigpen-carbohydrate binding is not likely due to metal chelation because pigpen binding to carbohydrate is insensitive to EDTA and 1, 10-phenanthroline, which would otherwise be expected to mimic the DTT effect. Our results suggest that DTT can interact with protein domains in the absence of cysteine residues, and that the biochemical reactivity of DTT is not necessarily one and the same with its assumed biochemical specificity.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Carbohydrate Metabolism
  • Cattle
  • Cloning, Molecular
  • Cysteine / metabolism
  • Cysteine / physiology
  • DNA-Binding Proteins
  • Disulfides / metabolism*
  • Dithiothreitol / pharmacology*
  • Edetic Acid / pharmacology
  • Enzyme Inhibitors / pharmacology*
  • Extracellular Matrix Proteins
  • Glycoproteins / chemistry
  • Lectins
  • Molecular Sequence Data
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / metabolism
  • Phenanthrolines / pharmacology
  • Plasmids / metabolism
  • Protein Conformation
  • Protein Structure, Tertiary
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sea Urchins
  • Sugar Alcohols / pharmacology
  • Sulfhydryl Compounds / metabolism*

Substances

  • DNA-Binding Proteins
  • Disulfides
  • Enzyme Inhibitors
  • Extracellular Matrix Proteins
  • Glycoproteins
  • Lectins
  • Nuclear Proteins
  • Phenanthrolines
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Sugar Alcohols
  • Sulfhydryl Compounds
  • echinonectin protein, Lytechinus variegatus
  • pigpen protein, bovine
  • threitol
  • Edetic Acid
  • Cysteine
  • Dithiothreitol
  • 1,10-phenanthroline