304 THE FORMATION AND STRUCTURE OF AN ADAPTIVE ENZYME, TRYPTOPHANASE, AND ITS SPECIFIC ANTI-ENZYME.* DORIS E. DOLBY, D. A. HALL AND F. C. HAPPOLD. From the Departments of Biochemistry and Medicine, School of Medicine, University of Leeds. Received for publication December 10, 1951. KtMMERLING (1943) prepared killed suspensions of Bacterium coli, Proteus X 19 and certain vibrios which possessed tryptophanase action. The techniques employed were those originally used by Happold and Hoyle (1934, 1935). He showed that the tryptophanase from Bact. coli could be inhibited by an anti-coli serum, and that the Proteus X 19 tryptophanase could be similarly inhibited by the serum of typhus patients which give a positive Weil Felix reaction. He suggested that the anti-tryptophanase produced in response to each species shows a separate species specificity, but there was little evidence for this. Dolby and Happold (1951), unaware of the wartime work of Kummerling, prepared an anti-tryptophanase to partially purified tryptophanase from Bact. coli cells. The work which is reported here is a continuation of that concerned with the purification and characterisation of the Bact. coli tryptophanase, which has been recently reviewed elsewhere (Happold, 1950). METHODS AND MATERLALS. Preparation of Protein Fractions from Cells of Bact. coli. Previous work had shown that it was possible to obtain a fraction rich in tryptophanase by extracting an acetone dried powder of Bact. coli cells with 1/4 saturated KCI solution for 2 hr. at 370 (10 ml. KCI solution per 100 mg. dried cells). Cellular debris was removed by centrifuging for 1 hr. at 3000 r.p.m. The supernatant was either used as such, or in cases where the high potassium content would have had undesirable effects, such as in injection into animals during immunisation experiments, was dialysed against 0-01 M sodium phosphate buffer pH 7*0 for 2 hr. For the experiments described below the cells have been grown for 16-18 hr. at 370 in three different media: (1) a broth based on a tryptic digest of casein, fortified by the addition of tryptophan (25 mg./l.) to produce a high concentration of tryptophanase; (2) a similar tryptic digest broth with the addition of 1 per cent glucose; the inclusion of carbohydrate inhibits the production ofthe enzyme; (3) a gelatine hydrolysate medium which gives good growth without the pro- duction of enzyme. The cells from these three types of culture were dried and protein fractions extracted as outlined above. The three preparations are referred to henceforth as " Tryptophanase," " Protein 1 " and " Protein 2." * Part of this work was presented to the 12th International Congress of Pure and Applied Chemistry, New York, September. 1951. TRYPTOPHANASE AND ITS ANTI-ENZYME 305 Dialysates of the three preparations, rendered isotonic with NaCl, were administered intravenously to rabbits in doses of 0-25, 0 5 and 0 5 ml. at intervals of one week. Antisera obtained by bleeding one week later, and designated Anti-T, Anti-G and Anti-Gel respectively, were not fractionated, but used whole. Examination of the amino-acid composition of various protein fractions. Protein samples (usually 5-10 mg.) were hydrolysed with 6 N HC1 in a sealed tube at 105° for 24-48 hr. Alkaline hydrolysis was with 5 N NaOH either in a sealed tube at 1050 for 6 hr., or under reflux for 24 hr. In certain experiments, 2 N baryta was used for hydrolysis for 3 hr. at 10 lb. per sq. in. pressure in an autoclave. The HCI was removed by repeated evaporation to dryness in vacuo, and the NaOH by electrolytic de-salting (3 hr. at a voltage of 120 v. to bring the current from 1*2 amp. to 20-30 m.amp.). Barium was removed by precipitation as sulphate. Chromatography was carried out on aqueous solutions of the hydrolysates; 10 jd. (containing 100 ,ug. N) was added to the paper. Two-way chromatograms were obtained by upward displacement (Williams and Kirby, 1948) on 25 cm. sheets of Whatman No. 4 paper, using phenol saturated with water or 63 per cent lutidine in one direction, and the alcoholic layer from a mixture of n-butanol, acetic acid and water (50:50:12) in the other. It was found that the best results in the shortest time could be obtained by running the phenol or lutidine for 6 hr. in an incubator at 300, while the butanol was run for 18 hr. at 180. The approximate concentrations of the individual amino-acids separated by the above procedure are assessed by visual appraisal of the intensity of the colours produced by treatment with ninhydrin. These intensities were estimated by comparison with colours produced by known quantities of the relevant amino-acid run under identical conditions. The data present in Table I are combinations of many results from both acid and alkaline hydrolvses, while those in Table IV are from alkaline hydrolyses alone. The latter, therefore, do not give a true indication of the relative concentrations of serine in the various preparations, since this amino-acid is partially destroyed during alkaline hydrolysis (cf. Nicholet and Shinn, 1941). In none of the chromatograms was any account taken of possible cystine content. The lower limit of observation is ofthe order of 2-3 ,ug., so that allowing for a similar variation in molecular weight between the range of amino-acids, any individual component present in a concentration of less than 0 3-0*4 per cent will not be recorded. This may explain the variety of results quoted in Table I, where only those amino-acids present at considerable concen- trations above the threshold were invariably discernible on the chromatograms. EXPERIMENTAL. Properties of the enzyme and anti-enzyme preparations. The activity of enzyme preparations was estimated by the normal method of indole production from tryptophan, and it was shown that the more purified the preparation, the more rapidly did the enzyme lose activity. Chromatograms of enzyme preparations are given in Table I. The amino-acids listed in the first column were invariably present in the hydrolysates, while those listed in the fourth column were present in some preparations but not invariably at concentrations 306 D. E. DOLBY, D. A. HALL AND F. C. HAPPOLD sufficiently high for observation. The marked instability of purified enzynme preparations handicaps the analysis of the purified enzvme. Aged tryptophanase can be divided into an insoluble protein fraction and a soluble, portion. Neither of these retains tryptophanase activity, and though it is difficult to determine slight changes in amino-acid composition in either of these fractions, almost all the alanine of the original enzyme preparation is segregated in the insoluble portion, and up to the present tryptophan has only been detected in the soluble supematant. The amino-acid composition of the two other protein fractions (Proteins 2 and 3) are also given in Table I. High concentrations of glutamic acid, aspartic acid, lysine and arginine which occur in the protein extracted from cells grown in the presence of glucose differentiate this protein from the active extract, as also does the absence of the aromatic amino-acids. The extract from the cells grown on gelatine is characterised by the invariable presence of hydroxyproline. TABLE I.-Amino-Acids Present in Protein Preparations: Composite Assessments Based on a Number of Chromatograms. Amino-acids Additional amino-acids invariably present. present in some preparations. r A'. f- 'I Amino-acids. Crude Crude trypto- Protein 1. Protein 2. trypto- Protein 1. Protein 2. phanase. phanase. Tryptophan + Phenylalanine +1C* Tyrosine ± Methionine * - * - + Arginine + + + Alanine . + * - * - + Serine *- + ± Glycine . + +F + a-amino butyric ±e + Proline +F * + -4- + Glutamic 4- + + * - * @ Aspartic ** + + Lysine + + ± Valine ± Leucine + + Isoleucine + + + Hydroxyproline * @ * @± Examination of the enzyme-antibody complex. Inactivation experiments fell into two groups: 1. The study of the period of inactivation during which the antigens are combining with antibodies. This was in the first instance effected by incubating the reactant mixtures for 2 hr. at 370, followed by 1 hr. at 00; in later experiments the reaction was allowed to proceed overnight at 00. The resulting precipitates were separated from the supematant by centrifuging. In the early experiments control tubes were set up using saline in place of antisera; however, this was TRYPTOPHANASE AND ITS ANTI-ENZYME 307 found to cause considerable inactivation of the enzyme on its own account (KCl caused less inactivation than NaCl), and in the later work normal rabbit serum was employed as a control, a procedure which resulted in a greater stabilisation or protection of the enzyme. 2. The measurement of the enzymic activity of the separated precipitates and supematants. This was carried out in 25 ml. Erlenmeyer flasks containing excess tryptophan, and crude pyridoxal phosphate dissolved in 0-013 M phosphate buffer, pH 7 0. The total volumes of the reactants were either 6 or 10 ml.; 1 ml. aliquots were removed after incubation at 37°, generally at 15-min. intervals. The indole was extracted with light petroleum, B.P. 40-60° treated with Ehrlich's reagent, and the depth of colour measured in a diffraction grating spectrophoto- meter at a wavelength of 560 mit. Preliminary experiments in the precipitation relationships between the antigens and antisera showed that equal volumes of antigen and antiserum left excess precipitins in the supernatant fluid. The capacity of the three types of sera, Anti-T, Anti-G and Anti-Gel, to inhibit tryptophanase action was tested. Active tryptophanase (1 ml.) was treated with 0*5 ml. Anti-T and the precipitate removed. No enzyme activity remained in the supernatant, while similar amounts of enzyme treated with equal and then double amounts of Anti-G and Anti-Gel sera showed no measurable loss of activity although considerable precipitation occurred.