Protein Protocols Protein Protocols

Cysteine Residues and Disulfide Bonds 597
89
Quantitation of Cysteine Residues
and Disulfide Bonds by Electrophoresis
Alastair Aitken and Michèle Learmonth
1. Introduction
Amino acid analysis quantifies the molar ratios of amino acids per mole of protein.
This generally gives a nonintegral result, yet clearly there are integral numbers of the
amino acids in each protein. A method was developed by Creighton (1) to count integral
numbers of amino acid residues, and it is particularly useful for the determination
of cysteine residues. Sulfhydryl and disulfide groups are of great structural, functional,
and biological importance in protein molecules. For example, the Cys sulfhydryl is
essential for the catalytic activity of some enzymes (e.g., thiol proteases) and the interconversion
of Cys SH to Cystine S—S is directly involved in the activity of protein
disulfide isomerase (2). The conformation of many proteins is stabilized by the presence
of disulfide bonds (3), and the formation of disulfide bonds is an important
posttranslational modification of secretory proteins (4).
Creighton’s method exploits the charge differences introduced by specific chemical
modifications of cysteine. A similar method was first used in the study of immunoglobulins
by Feinstein in 1966 (5). Cys residues may be reacted with iodoacetic acid, which
introduces acidic carboxymethyl (—O 2CCH 2S—) groups, or with iodoacetamide,
which introduces the neutral carboxyamidomethyl (H 2NCOCH 2S—) groups. The reaction
with either reagent is essentially irreversible, thereby producing a stable product
for analysis. Using a varying ratio of iodoacetamide/iodoacetate, these acidic and neutral
agents will compete for the available cysteines, and a spectrum of fully modified
protein molecules having 0,1,2, … n acidic carboxymethyl residues per molecule is
produced (where n is the number of cysteine residues in the protein). These species will
have, correspondingly, n, n – 1, n – 2, … 0 neutral carboxyamidomethyl groups. These
species may then be separated by electrophoresis, isoelectric focusing, or by a combination
of both (1,6,7). The examples of the analysis of the cysteine residues in bovine
pancreatic trypsin inhibitor and ovotransferrin are shown in Fig. 1.
Creighton used a low-pH discontinuous system (1). Takahasi and Hirose recommend
a high-pH system (6), whereas Stan-Lotter and Bragg used the Laemmli electrophoresis
system followed by isoelectric focusing (7). It may therefore be necessary to
carry out preliminary experiments to find the best separation conditions for the protein
under analysis. The commonly used methods are given below.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
597