The Composition, Structure and Reactivity of Proteins. A by P. Alexander, R. J. Block

By P. Alexander, R. J. Block

The Composition, constitution and Reactivity of Proteins

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Extra info for The Composition, Structure and Reactivity of Proteins. A Laboratory Manual of Analytical Methods of Protein Chemistry (Including Polypeptides)

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Then 2 ml more of ninhydrin are added and the solution is gently boiled 25 min longer. Aeration is continued throughout. The bisulphite solution which now contains the acetaldehyde is diluted to 10 ml. Colour development. Pipette 1 ml of bisulphite solution which contains acetaldehyde equivalent to 0·002-0·008 mg of alanine into a reading tube. 44 ANALYTICAL METHODS OF PROTEIN CHEMISTRY Add 1 drop of copper sulphate, 10 ml of concentrated H 2S04 (cool to room temperature), and approximately 50 mg of p-hydroxydiphenyl.

Apparatus The lactic acid apparatus of Friedemann and Kendall (1929) or any similar equipment is used. The acetaldehyde is collected in bisulphite which is kept in an ice bath. Method. Add to the oxidizing flask the following reagents: 1-5 ml of alanine solution (0·05-0·10 mg of alanine), 2 ml of buffer, and 2 ml of ninhydrin solution. The solution is boiled for 45-50 min during which time the acetaldehyde is aerated into the bisulphate with a gentle stream of air. Then 2 ml more of ninhydrin are added and the solution is gently boiled 25 min longer.

44 ANALYTICAL METHODS OF PROTEIN CHEMISTRY Add 1 drop of copper sulphate, 10 ml of concentrated H 2S04 (cool to room temperature), and approximately 50 mg of p-hydroxydiphenyl. Stopper, mix from time to time and allow the colour to develop overnight. The colour is read using filter 540 m,u against water. 11. Hydroxyproline (Neuman and Logan, 1950) Reagents 0·01 MCuS04. 2·5NNaOH. 6% vjv H 20 2. 3·ONH2S04· 5 % wjv of recrystallized p-dimethylaminobenzaldehyde in n-propanol. Method. Standards in duplicate: 1 ml containing 5,ug of hydroxyproline, 1 ml containing 10 ,ug of hydroxyproline, 1 ml containing 15 ,ug of hydroxyproline.

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