This experiment describes the cyanogen bromide cleavage process of calmodulin. This experiment is from the Experimental Guide for Protein Purification and Identification, by Zhu Houzhu.

Operation method

Cyanogen bromide cleavage assay of calmodulin

Materials and Instruments

Formic acid Pure calmodulin Cyanogen bromide Acetonitrile

Move

makings

Formic acid (the highest grade commercially available (e.g. Aldrich Chemical Co., Inc.))

Pure calmodulin (dry powder; no salt (0.1~1 mg))

Cyanogen bromide (CNBr) (crystal) (e.g. AldrichChemicalCo., Inc.)

Acetonitrile (e.g. Burdick & Jackson or J,T. Baker)

Operating Procedures

This operating procedure has been modified from the literature of Gross and Witkop (1962).

1) Dilute formic acid with water to a final concentration of 70% (w/v). For >99% formic acid, use 2.1 ml formic acid and 0.9 ml water. For 88% formic acid, use 2.lml formic acid and 0.54 ml water.

2) Dissolve calmodulin in 70% formic acid solution at a final concentration of 1 mg/ml on ice.

3) Weigh 1.5-ml capped polypropylene centrifuge tube. Wearing gloves and in a fume hood, open the CNBr bottle and transfer a small pellet of crystals to a pre-weighed centrifuge tube. Close the tube and CNBr bottle. Weigh the CNBr-filled tube and subtract the weight of the tube to calculate the weight of CNBr.

4) Dissolve CNBr in acetonitrile to a final concentration of 6 md/L (0.64 g/ml). Remember to wear gloves and open the tube only in a fume hood.

5) Add one aliquot of CNBr solution to the calmodulin. The amount of CNBr in this aliquot should exceed 400 times the total molar amount of methionine residues expected in calmodulin.

6) Cover the tube tightly and mix thoroughly. Wrap the tube in foil to protect it from light. Incubate the reaction mixture in a fume hood for 22 h at room temperature.

7) Add an equal volume of purified water to the reaction mixture. The sample was centrifuged under vacuum to dryness. The sample was then redissolved in 0.1% (w/v) trifluoroacetic acid for HPLC or mass spectrometry analysis.

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