A Colorimetric Method for the Determination of the Proteolytic Activity of Duodenal Juice

The coupling of diazotized aryl amines with proteins in alkaline solution yield chromophoric protein derivatives.Digestion of a solution of such azoproteins with proteolytic enzymes results in the formation of colored components soluble in trichloroacetic acid.The intensity of the color in the trichloroacetic acid filtrate of the digested substrate is a function of the proteolytic activity of the enzyme solution and serves as the basis for the method here described.EXPERIMENTAL Preparation of Sulfanilamide-Axocasein--Solution A, 50 gm. of reprecipitated, fat-free casein are dissolved with stirring in 1 liter of water containing 10 gm. of NaHC03.Solution B, 5.0 gm. of sulfanilamide are dissolved in 200 ml. of Hz0 containing 6.0 ml. of 5.0 N NaOH.2.20 gm. of NaNOz are added.To the stirred solution 18.0 ml. of 5 N HCl are added and stirring is continued for 2 minutes.18.0 ml. of 5 N NaOH are then added; the solution is stirred and added at once, with stirring, to Solution A.The azoprotein is precipitated by acidification to pH 4.5, washed with water and alcohol, and air-dried.The azocasein is a red-orange compound with an absorption maximum at 440 mp (Fig. 1).Substrate Solution-A stock solution of the substrate containing 25 mg. of azocasein and 5 mg. of sodium bicarbonate per ml. is prepared by dissolving 2.50 gm. of azoprotein in 50 ml. of 1.0 per cent NaHC03 at 60" with stirring.The pH is adjusted to 8.3 and the solution diluted to 100 ml. with distilled HzO.The stock solution is stored at 0".Duodenal Juice-The specimens are centrifuged at about 1500 R.P.M. for lominutes and the sample taken from the relatively homogeneous middle layer. 1 ml. of sample is diluted to 100 ml. with bicarbonate buffer (5 mg. per ml.), pH 8.3. ProcedureEach determination is set up in duplicate.The flask containing the substrate solution and a rack with the proper number of tubes are placed in a 501