Phenylmethylsulfonyl fluoride is a serine protease inhibitor that irreversibly inhibits serine proteases by sulfonylation of the serine residue in the active site of the protease. It is widely used in protocols for the isolation of proteins.

Properties

Phenylmethylsulfonyl fluoride is a crystalline solid of low toxicity that is a potent, irreversible inhibitor of chymotrypsin and other serine proteinases . Aqueous solutions of PHENYLMETHYLSULFONYL FLUORIDE undergo slow hydrolysis, but it is reasonably stable in anhydrous alcohol. When used to minimize protein degradation during isolation procedures, it is added to buffer solutions prior to use by 100-fold dilution from a stock 50 mM solution in isopropanol. The product of reaction with chymotrypsin is the phenylmethylsulfonyl ester of the active site serine.Although this ester is quite stable, it is possible to regenerate the active enzyme by incubation under acidic conditions (pH 2) for several hours. It is also possible, under alkaline conditions, to convert the product to anhydrochymotrypsin in which the active site serine residue becomes a dehydroalanine residue.

Biological Role

Phenylmethylsulfonyl fluoride has biological roles as serine proteinase inhibitor.

Applications

In biochemistry, Phenylmethylsulfonyl fluoride (PHENYLMETHYLSULFONYL FLUORIDE) is a serine protease inhibitor (serine hydrolase inactivator) commonly used in the preparation of cell lysates. PHENYLMETHYLSULFONYL FLUORIDE does not inactivate all serine proteases. The effective concentration of PHENYLMETHYLSULFONYL FLUORIDE is between 0.1 - 1 mM. The half-life is short in aqueous solutions (110 min at pH 7, 55 min at pH 7.5, and 35 min at pH 8, all at 25 °C).Stock solutions are usually made up in anhydrous ethanol, isopropanol, or corn oil and diluted immediately before use.

Phenylmethylsulfonyl fluoride binds specifically to the active site serine residue in serine hydrolases. It does not bind to any other serine residues in the protein. This is a result of the hyperactivity of that serine residue caused by the specific environmental conditions in the enzyme's active site (catalytic triad). Because PHENYLMETHYLSULFONYL FLUORIDE binds covalently to the enzyme, the complex can be viewed by X-ray crystallography; it can therefore be used as a chemical label to identify an essential active site serine in an enzyme.

Enzyme(active)Ser-O-H + F-SO2CH2C6H5 → EnzymeSer-O-SO2CH2C6H5 + HF

Serine protease + PHENYLMETHYLSULFONYL FLUORIDE → Irreversible enzyme-PMS complex + HF

The median lethal dose is less than 500 mg/kg(acetylcholine esterase inactivator!). PHENYLMETHYLSULFONYL FLUORIDE should be handled only inside a fume hood and while wearing gloves. DMSO is sometimes recommended as solvent for stock solutions, but should not be used as it makes intact skin permeable for PHENYLMETHYLSULFONYL FLUORIDE.