Iodoacetamide and iodoacetate are two of the most common reagents used for chemically modifying thiol groups irreversibly. They have a long history, dating back to 1874 when it was reported that bromoacetate injected subcutaneously into frogs killed them, as a result of progressive respiratory and cardiac depression; iodoacetate was subsequently found to be more reactive. These studies were important in elucidating the mechanism of glycolysis, which led to the realization that iodoacetate and other such compounds acted by reacting with the thiol groups of proteins, in this case at the active site of the enzyme glyceraldehyde-3-phosphate dehydrogenase. Iodoacetamide became popular because it can cross cell membranes much more readily than can iodoacetate and therefore is more potent with cells and organs.
Both iodoacetamide and iodoacetate react rapidly and specifically with the ionized form of thiol groups, to generate very stable adducts:
These adducts are commonly known as the carboxamidomethyl and carboxymethyl, respectively. They are stable to conditions routinely used to hydrolyze proteins to their substituent amino acids, except that the carboxamidomethyl group gets hydrolyzed to the carboxymethyl (see Amino Acid Analysis).
The reaction with iodoacetamide or iodoacetate occurs by the thiolate anion attacking and displacing the iodine atom in a nucleophilic reaction. Consequently, the rate of the reaction increases with increasing pH, up to just beyond the pKa of the thiol group, which typically is about 9 for most biological substances. The pH dependence of the rate of the reaction can be used to measure the apparent pKa value of the thiol group, although there are exceptional cases in catalytic proteins where the results are misleading, presumably because the protein modifies the energy of the transition state for the reaction (1). The reaction occurs rapidly with thiol groups. The second-order rate constant for reaction at 25°C of iodoacetamide with a fully ionized cysteine thiol group, with a pKa of about 8.7, is approximately 25 s-1M-1. Consequently, the half-time for modification of such a thiol group upon adding 0.1 M iodoacetamide is only 0.3 sec. Iodoacetate reacts about three times less rapidly than does iodoacetamide.
It is important to realize that protons are released by the reaction. Frequently, high concentrations of a thiol reagent are used to reduce all cysteine residues, and then a large excess of iodoacetate or iodoacetamide is added to react with both the protein and the reagent. In this case, the pH will drop and the rate of the reaction will slow, and even stop before completion, unless very high concentrations of a buffer are present.
The two reagents differ only in one being an amide, and neutral, and the other an acid, and negatively charged. This charge difference is useful for counting residues of thiol groups in a protein. Mixtures of the two reagents are reacted with a protein containing N thiol groups, to produce a spectrum of molecules with net charge differences of 0 to N. If this spectrum is resolved by electrophoresis, isoelectric focusing, ion-exchange chromatography, or other techniques dependent only upon the charge of the protein, the number of species with different charges present can be counted, giving the integer value of N.
Both iodoacetamide and iodoacetate also react with amino groups, but at a lower rate, and primarily at very alkaline pH values, where the reactive nonionized amino groups are present. Both lysine and histidine residues can be modified in this way. The reaction of ribonuclease A, which has no free thiol groups, with the two histidine residues at its active site, each reacting with different N atoms, was important in demonstrating the specificity of the interactions that could take place in enzyme-active sites (2). They can also react with the sulfur atom of methionine residues to generate the positively charged sulfonium salt, but at a much lower rate (3). This rate is essentially independent of pH, however, so it can become the predominant modification reaction of a protein at very low pH values. The reactions of iodoacetamide and iodoacetic acid can be used in diagonal methods to isolate specifically cysteine- and methionine-containing peptides.
Many variants of iodoacetate and iodoacetamide have been devised by adding further groups with absorbance and fluorescence properties that serve as reporters of structure in proteins.
