The Estimation of Histidine

The estimation of arginine, histidine and lysine in small amounts of protein by the silver precipitation method has been satisfactorily employed by Miller, Mazur, Plimmer, and others. However, Abderhalden and Siebel were only successful in the determination of arginine and lysine. The latter investigators reported large losses of histidine during the purification procedures in which HgSO4 and CuCO3 are employed. It has been recognized that histidine is the most difficult of the basic amino acids to determine accurately and we take this occasion to present some details of the modifications of the 1934 procedure. Histidine is precipitated from the protein hydrolysate at pH 7.4 with AgNO3 and Ba(OH)2. The precipitate is centrifuged, washed and decomposed with H2S after suspending in dilute H2SO4 (cf. for apparatus). The Ag2S is removed and the filtrate and washings are concentrated in vacuo (cf.). The filtrate is adjusted to pH 3.5-4.5 with Ba(OH)2, and after removal of the BaSO4, the histidine solution is concentrated to approximately 20 cc. and decolorized with charcoal. The precipitate is filtered, thoroughly washed with hot H2O and the filtrate is diluted to volume (100 cc). Histidine may be determined directly as follows. (a) Colorimetric (Kapeller-Adler). 2 cc. portions (containing 0.5-1.0 mg. of histidine per cc.) of the sparkling histidine sulfate solution are pipetted into 6 test tubes graduated at 10 cc. Into 6 other similar tubes, an equal quantity of histidine standard is placed. (For best results, standard and unknown should contain approximately the same amounts of histidine.) The 12 tubes are set up alternately and an excess of Br2 in dilute acetic acid (cf.) is added. The bromination is allowed to proceed for 10 minutes, care being observed that all the solutions are colored yellow at the end of this period. The excess Br2 is removed by a drop of As2 O3, and 2 cc. of NH4 0H reagent (cf.) per tube are added immediately. The contents of the tubes are mixed and they are placed in mildly boiling H2O for 5 minutes. At the end of this period, the solutions are cooled in ice for 10 minutes and diluted to volume with NH4OH reagent. The contents of each group of test tubes are mixed and the colors matched in the usual fashion. (b) Gravimetric (nitranilic acid). In 1936, Town claimed that nitranilic acid (2,5-dihydroxy-3,6-dinitroquinone) specifically precipitates glycine from an ethanolic solution of the products of protein Itydrolysis. Stein found that the amount of glycine in gelatin estimated by Town's method was greater than that determined by Bergmann and Fox and that this discrepancy could be explained by the partial precipitation of the basic amino acids along with glycine. We have found that nitranilic acid can be used successfully for the isolation of histidine as follows: The remainder of the histidine solution (cf. a) is concentrated in vacuo (pH 3.5-4.0) and an excess of nitranilic acid (freshly dissolved) in H2 O, (CH3)2 CO, or CH3 OH is added. The finely crystalline, yellow precipitate is filtered off, washed and dried at 110°. The gravimetric and colorimetric estimations check very closely, on analysis of 15 brain proteins, if the factor 0.403 × the weight of the yellow precipitate is employed. Further work along this line is now in progress. In certain instances, further purification of the histidine fraction is desirable before estimation of the amino acid. In such cases, the histidine sulfate solution is concentrated to approximately 120 cc. and an excess of Denige's reagent (cf.) is added. The suspension is centrifuged for 20-30 minutes and the filtrate is discarded. The precipitate is suspended in H2O and decomposed with H2S. After removal of the HgS and H2S, the histidine solution is adjusted to pH 5-6 with Ba(OH)2. BaSO4 is removed and washed. The filtrate is boiled and it is made alkaline to litmus by the addition of minimal quantities of BaCO3. An excess of CuSO4 Results indicating the probable errors of the method, which are based on more than 10 experiments, each starting with 0.1355 gm. of histidine hydrochloride hydrate are given: