Oxidation of proteins with peroxyformic acid is a simple and effective method of partially denaturing proteins and stabilizing sulfur-containing amino acids. Performic acid oxidation quantitatively converts cysteine residues to cystine and methionine residues to methionine sulfone. These residues are stable to acid hydrolysis. This experiment was derived from the Laboratory Guide for Protein Purification and Characterization by Houzhu Zhu.

Operation method

Performic acid oxidation experiments on proteins

Materials and Instruments

Hydrogen peroxide Formic acid
Ice Water bath

Move

Materials and equipment

Hydrogen peroxide (fresh) (30%)

Formic acid (highest purity grade available)

Ice/water bath

Operating Procedures

This procedure has been modified from the method of Hirs (1967).

1) Prepare the formic acid reagent by mixing hydrogen peroxide with formic acid in a ratio of 1:19 (v/v) (e.g., 0.5 ml of hydrogen peroxide and 9.5 ml of formic acid). The mixture must be allowed to stand for a full 2 h at room temperature (25°C) in a tightly closed container.

2) Place the formic acid reagent in an ice/water bath. Dissolve the protein to be oxidized in formic acid (a concentration of lmg/ml is usually sufficient). Place the dissolved protein sample in an ice/water bath. Let both the protein sample and the formic acid reagent stand in the ice/water bath for 30 min until they are cold.
Note: Ice alone will not cool the tubes or the ice/water bath.

3) Add 2 to 3 times (by volume) the excess of formic acid reagent to the protein sample. Incubate the reaction mixture for 2.5 h in an ice/water bath.

4) Add a small amount (10% by volume) of pure water (deionized or distilled) to the reaction mixture and mix thoroughly. Remove formic acid, performic acid and water by evaporation in a vacuum centrifuge to dryness. Redissolve the protein sample in water and redry in a vacuum centrifuge.
Note: When drying a protein sample from an organic acid such as methylhydrazine, it will not appear as a white or crystalline substance, but rather as a clear "glassy" form. Therefore, do not expect to be able to easily see the dried protein at the bottom of the tube. If a clean test tube is illuminated with a lamp, the dried proteins can be observed by the change in transmitted light.

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