Die colorimetrische und enzymatische Bestimmung des Histidins

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Abstract
Diazo soln. 1 is prepared by adding 0.5 g. p-mono-chloroaniline to 5 ml. conc. HCl and diluting the mixture to 200 ml. with water. The diazo soln. 2 is an 0.5% soln. of NaN02 in water. The diazo reagent is prepared by mixing 2 parts soln. 1 with 1 part soln. 2. One ml. of the histidine soln. is placed in a 50 ml. flask, 5 ml. M/20 Na2CO3 soln. added and then 0.5 ml. of the diazo reagent is added drop by drop. The flask is placed in ice water 3 min., and the azo pigment extracted with 10 ml. n-butyl alcohol, and the cone, of pigment in the alcohol detd. in the step-photometer with filter S 45. The cone, of histidine may then be detd. by means of a standard reference curve, the lower limit of the method being 67/ml. soln. To determine histidine by the enzymatic method, histidase is prepared by grinding rat livers with double their wt. of sand, stirring the mixture 5 min. with 4 vols. M/15 phosphate buffer, pH 8, and cen-trifugalizing. Five ml. of the supernatant histidase soln. are added to 2 ml. of about N/10 histidine soln. and 13 ml. of phosphate buffer. One ml. of toluene is placed on the mixture, which is incubated at 38[degree] for 24 hrs. The mixture is made strongly alkaline and the NH3 liberated detd. by the aeration and titration method of Folin. Experience has shown that 10% must be added to the amt. of NH3 found to obtain 2/3 of the histidine N. The other 1/3 of histidine N is not liberated as NH3.

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