The estimation of arginine

Abstract

Arginine has been estimated by precipitation as the picrate and flavianate complexes, enzymically by the action of arginase followed by the estimation of the urea formed, microbiologically and colorimetrically (cf. Block & Boiling, 1947). The colorimetric method developed by Sakaguchi (1925a, b) has been most widely used. This method relies upon the formation of a red-coloured complex produced by arginine and other monosubstituted guanido compounds in the presence of a-naphthol and alkaline hypobromite or hypochlorite. Although this method will qualitatively detect small quantities of arginine, it is unreliable quantitatively, as evidenced by the large number of modifications that have been suggested, and which have been reviewed recently by G6mez & Marenzi (1953). A colorimetric method for the estimation of both mono-and di-substituted guanidines was introduced by Eggleton, Elsden & Gough (1943). These compounds give a red-coloured complex with diacetyl and a-naphthol in alkaline solution. The authors found that monosubstituted guanidines such as arginine and glycocyamine produced only one-ninth of the colour given by asymmetrically disubstituted guanidines such as creatine. This observation has been confirmed by Ennor & Stocken (1953) and by Roche, Thoai & Hatt (1954). Although this method is less sensitive than the Sakaguchi method, it is simpler and more reliable. This paper describes a modification of the above reaction. When the colour is developed in the presence of n-propanol and an increased concentration of x-naphthol, the sensitivity of the method for monosubstituted guanidines is equal to that previously obtained with disubstituted guanidines. Thus it is possible to estimate arginine in amounts ranging from 10 to 100 ,ug. EXPERIMENTAL Materials n-Propanol. n-Propanol was purified by distillation after refluxing over NaOH and Al powder. a-Naphthol. The commercial material was light brown in colour and was purified by two steam-distillations from 2N-H2SO4. It was then dried in vacuo at 60°, and dissolved in n-propanol to give a 25 % (w/v) solution, and stored in a dark bottle at-20°. Stock diacetyl solution. A stock diacetyl solution (about 1 %) was prepared from dimethylglyoxime as described by Walpole (1911). Arginine. Arginine hydrochloride was recrystallized from 0-4N-HC1 (cf. Greenberg, 1951), and its purity was checked by N analysis. Other guanido compounds. Guanidine (A.R.) was used. Creatine was recrystallized as described by Hunter (1928). Glycocyamine (guanidoacetic acid) was recrystallized from water. N-Ethylguanidoacetic acid (negmine) was kindly supplied by Dr M. D. Armstrong (see Ennor, Rosenberg & Armstrong, 1955). a-Guanidopropionic acid, ,-guanidopropionic acid and ,-guanidoethylsulphonic acid (taurocyamine) were prepared by guanylation with S-ethylthiourea of alanine, ,-alanine and taurine respectively, as described by Brand & Brand (1942) for the preparation of glycocyamine. 1:2-Diguanidoethane, 1-guanidoethane and 2-guanidoethanol were prepared from ethylenediamine, ethylamine and et...

Characterization of 3-Guanidinopropionate Amidinohydrolase fromPseudomonas aeruginosaand a Comparative Study with 4-Guanidinobutyrate Amidinohydrolase from anotherPseudomonas

“…Urea was determined by the color reaction with diacetylmonoxime and antipyrine according to the procedure of Prescott and Jones 7); color development was carried out at 100°C for 40 min in the dark. 3-Guanidinopropionate and 4-guanidinobutyrate were determined colorimetrically by use of a-naphthol and 2,3-butanedione according to Rosenberg et a1 8 ) The 'protein concentration of the crude extract or ammonium sulfate fraction was measured by the method of Lowry et a1 9 ) with bovine serum albumin as a standard. For more extensively purified preparations, the protein concentration was estimated from absorbance at 280 nm using an E (I %) value of 10.0.…”

Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

“…Determination of the arginine content of the cell lysate was carried out as already described (29). Briefly, equal volumes of test samples and 3 N NaOH were mixed, a developing solution containing 5% (wt vol Ϫ1 ) ␣-naphthol and 2.5% (vol vol Ϫ1 ) diacetyl in water was added, and the mixture was incubated for 30 min at room temperature.…”

Taurocyamine-utilizing Mutant ofPseudomonas aeruginosawith Altered Induction of 3-Guanidinopropionate Amidinohydrolase

“…Guanidino compounds were assayed colorimetrically by the method of Rosenberg et al 10 ) Urea was assayed with diacetyl monoxime and antipyrine by the method of Prescott and Jones. 11) Protein in th~cell extracts.…”