Slow Protein Hydrolysis in the Presence of 2,4-Dinitrophenylhydrazine

Abstract

IN our search for reactive keto groups in the protein molecule, we found that 2,4-dinitrophenylhydrazine could be coupled with the protein framework to a surprisingly great extent (one molecule of the hydrazine to about six amino-acid residues). In a typical experiment, 100 gm. casein ("nach Hammarsten") was boiled with 1,000 c.c. 0·3 N hydrochloric acid and 10 gm. 2,4-dinitrophenylhydrazine. From time to time 4 c.c. samples of the mixture were removed, precipitated with 5 c.c. 10 per cent trichloroacetic acid, centrifuged, and 2 c.c. of the supernatant liquid and the whole of the precipitate dissolved in N sodium hydroxide, each to a total volume of 50 c.c. The extinction coefficients of these deep red solutions were measured in a Zeiss colorimeter (green filter, 0·5 cm. cuvette). When the hydrazine is not coupled, the red colour fades immediately. The intensity of the permanent colour is found to be proportional to the amount of hydrazine bound in a similar manner to that occurring in hydrazones¹.