124 N. L. BLUMSOM AND J. BADDILEY 1961 Baddiley, J., Buchanan, J. G. & Carss, B. (1957a). J. chem. Kornfeld, S. & Glaser, L. (1960). Biochim. biophy8. Acta, Soc. p. 1869. 42, 548. Baddiley, J., Buchanan, J. G. & Carss, B. (1957b). J. Kowalsky, A. & Koshland, D. E. (1956). Biochim. biophys. chem. Soc. p. 4138. Acta, 22, 575. Baddiley, J., Buchanan, J. G., Handschumacher, R. E. & Kuehl, F. A., Clark, R. L., Bishop, M. N., Flynn, E. H. & Prescott, J. F. (1956). J. chem. Soc. p. 2818. Folkers, K. (1949). J. Amer. chem. Soc. 71, 1445. Borenfreund, E. & Dische, Z. (1957). Arch. Biochem. Kwapinski, J. & Merkel, M. (1957). Bull. Acad. polon. Sci., Biophy&. 67, 239. Ser. sci. biol., 5, 335. Buchanan, J. G. (1951). Nature, Lond., 168, 1091. Manson,L. A. & Lampen, J. 0. (1951). J. biol. Chem. 191,87. Buchanan, J. G., Dekker, C. A. & Long, A. G. (1950). Markham, R. & Smith, J. D. (1949). Biochem. J. 45, 294. J. chem. Soc. p. 3102. Markham, R. & Smith, J. D. (1952). Biochem. J. 52, 552. Buchanan, J. G., Lynch, V. H., Benson, A. A., Bradley, Maxwell, E. S. (1956). J. Amer. chem. Soc. 78, 1074. D. F. & Calvin, M. (1953). J. biol. Chem. 203, 935. Michelson, A. M. & Todd, A. R. (1956). J. chem. Soc. Cabib, E. & Leloir, L. F. (1954). J. biol. Chem. 206, 779. p. 3459. Chambers, R. W. & Moffatt, J. G. (1958). J. Amer. chem. Moffatt, J. 0. & Khorana, H. G. (1958). J. Amer. chem. Soc. 80, 3752. Soc. 80, 3756. Colowick, S. (1938). J. biol. Chem. 124, 557. Okazaki, R. (1960). Biochim. biophy8. Acta, 44, 478. Consden, R. & Stanier, W. M. (1952). Nature, Lond., 169, Paladini, A. C. & Leloir, L. F. (1952). Biochem. J. 51, 426. 783. Park, J. T. & Johnson, M. J. (1949). J. biol. Chem. 181, Dische, Z. & Shettles, L. B. (1948). J. biol. Chem. 175, 595. 149. Dixon, J. S. & Lipkin, D. (1954). Analyt. Chem. 26, 1092. Partridge, S. M. (1948). Biochem. J. 42, 238. Fiske, C. H. & Subbarow, Y. (1925). J. biol. Chem. 66, 375. Pazur, J. H. & Shuey, E. W. (1960). J. Amer. chem. Soc. Frahn, J. L. & Mills, J. A. (1959). Au8t. J. Chem. 12, 65. 82, 5009. Fried, J. & Titus, E. (1948). J. Amer. chem. Soc. 70, 3615. Pontis, H. G. & Blumsom, N. L. (1958). Biochim. biophys. Fried, J., Walz, D. E. & Wintersteiner, 0. (1946). J. Amer. Acta, 27, 618. chem. Soc. 68, 2746. Racker, E. (1947). J. biol. Chem. 167, 843. Ginsburg, V. (1960). J. biol. Chem. 235, 2196. Reindel, F. & Hoppe, W. (1954). Chem. Ber. 87, 1103. Hanes, C. S. & Isherwood, F. A. (1949). Nature, Loud., 164, Staveley, H. E. & Fried, J. (1949). J. Amer. chem. Soc. 71, 1107. 135. Heppel, L. A. & Hilmoe, R. J. (1951). J. biol. Chem. 188, Strominger, J. L. & Scott, S. S. (1959). Biochim. biophys. 665. Acta, 35, 552. Hershey, A. D., Dixon, J. & Chase, M. (1953). J. gen. Stumpf, P. K. (1947). J. biol. Chem. 169, 367. Physiol. 36, 777. Trevelyan, W. E., Procter, D. P. & Harrison, J. S. (1950). Jeanes, A., Wise, C. S. & Dimler, R. J. (1951). Analyt. Nature, Lond., 166, 444. Chem. 23, 415. Woodward, R. B. (1957). Fe8tschrift Prof. Dr Arthur Stoll, Kalckar, H. M. & Maxwell, E. S. (1956). Biochim. biophy8. p. 524. Basel: Birkhauser. Acta, 22, 588. Wyatt, G. R. (1951). Biochem. J. 48, 584.

Biocl&em. J. (1961) 81, 124 The Ribitol Teichoic Acid from Lactobacillus arabinosus Walls: Isolation and Structure of Ribitol Glucosides

BY A. R. ARCHIBALD,* J. BADDILEY AND J. G. BUCHANAN Department of Chemi8try, King'8 College, Univer8ity of Durham, Newca8tle upon Tyne (Received 10 February 1961) It has been known for some time that consider- graphy, suggested that this compound is a polymer able amounts of a ribitol teichoic acid occur in the in which ribitol units are joinled together through wals of Lactobacillus arabinomu8 17-5 (Baddiley, phosphodiester linkages, and that glucosyl and Buchanan & Greenberg, 1957; Baddiley, Buchanan alanine ester groups are also present (Armstrong & Carss, 1958; Armstrong, Baddiley, Buchanan & et al. 1958b). It is clear that this teichoic acid Carss, 1958a; Armstrong, Baddiley, Buchanan, resembles the ribitol teichoic acid in the walls of Carss & Greenberg, 1958b). Preliminary studies, BaciUu8 subtili8 (Armstrong, Baddiley & Buchanan, carried out largely with the aid of paper chromato- 1960, 1961), but several differences in the detailed structure of the two polymers were observed in the * Frank Schon Fellow. preliminary work. Hydrolysis with acid and alkali Vol. 81 RIBITOL TEICHOIC ACID FROM L. ARABINOSUS 125 revealed that, whereas the compound from B. glucose, 20 g.; potassium dihydrogen phosphate, 4-5 g.; subtili8 is a regular polymer in which 4-0-(f-D-glu- sodium hydroxide, 1-04 g.; inorganic salts (Barton-Wright, copyranosyl)-D-ribitol units are joined through 1946), 5 ml.; distilled water, 100 ml. groups, that from L. arabinosuw The cells (about 80g. wet wt.) were harvested in a phosphodiester refrigerated Sharples centrifuge and washed with 0.95% contains mono- and di-glucosylribitol units. Pre- sodium chloride solution at 00. They were treated (30 sec.) parations from some batches of micro-organisms in a blender with successive portions of acetone, ethanol also contain ribitol units which bear no sugar. and ether (15 vol. of each) at room temperature. Moreover, whereas the glycosidic linkages in the Fat-free cells (160 g.) were extracted by blending for teichoic acid from B. subtilta have the P-configura- 1 min. with 10% trichloroacetic acid (600 ml.) at 20. The tion, those in the L. arabinosWu polymer probably residue, after removal of the supernatant solution by have the a-configuration. centrifuging, was extracted twice in a similar manner. The A more detailed investigation has now been combined extracts were filtered through Celite. To the carried out on the ribitol teichoic acid from L. clear yellow solution (1.5 1.) cold ethanol (2 1.) was added with stirring and the mixture stored at 00 for 24 hr., when arabinosus, and the earlier conclusions have been the resultant yellow-white precipitate was removed by substantiated. In this paper the isolation and centrifuging and redissolved in cold 10% trichloroacetic structure of ribitol mono- and di-glucosides from acid (120 ml.). After removal of insoluble material, cold hydrolysates of the teichoic acid is described. ethanol (150 ml.) was added and, after 24 hr. at 00, the precipitated teichoic acid was removed by centrifuging and dried with ethanol and ether. Yield, 667 mg. of powder EXPERIMENTAL composed largely of ribitol teichoic acid, together with some glycerol teichoic acid and nucleic acid. Material&. fl-Glucosidase was kindly provided by Dr The residues from the above extraction were resuspended W. J. Whelan; crude yeast hexokinase was a gift from in 10% trichloroacetic acid (700 ml.) and blended for 1 mi. Sigma Chemical Co., St Louis, Mo., U.S.A.; calf intestinal at 00. The suspension was then stirred at 00 for 24 hr. and phosphomonoesterase was purchased from Sigma Chemical again blended for 1 min. before centrifuging. The resulting Co.; DL-glyceric acid, adenosine triphosphate and reduced residue was stirred with 10% trichloroacetic acid (300 ml.) diphosphopyridine nucleotide were purchased from L. and again centrifuged, the combined solutions then being Light and Co. Ltd., Colnbrook, Bucks.; Glucostat reagent filtered through Celite and mixed with 1.5 vol. of cold was purchased from Worthington Biochemical Corp., ethanol. After 24 hr. at 00 the precipitated material was Freehold, N.J., U.S.A.; prostatic phosphatase was pre- collected as before. At this stage the material contained a pared from human prostate glands; L. arabinosus 17-5 was large amount of nucleic acid, much of which was removed grown from a culture supplied by Dr D. E. Hughes. by extraction of the teichoic acid with ice-cold distilled water (100 ml.). The insoluble material (416 mg.) was Methods removed by centrifuging and teichoic acid (1-316 g.) was Paper chromatography. Paper chromatography was obtained by precipitation with ethanol. This material con- carried out on Whatman no. 1, 4, and 3 mM paper that had tained ribitol teichoic acid and a little glycerol teichoic acid been washed with 2 N-acetic acid and then water. The and nucleic acid. The debris, which still contained teichoic following solvent systems were used: A, propan-l-ol- acid, was not extracted further. ammonia (sp.gr. 0-88)-water (6:3:1) (Hanes & Isherwood, Isolation of teichoic acid from Lactobacillus arabinosus 1949); B, butan-l-ol-ethanol-water-ammonia (sp.gr. 0.88) walls. The cells were grown for 16 hr. as described above (40:10:49:1) (organic phase) (Hirst, Hough & Jones, and, after washing with cold 0-95% sodium chloride, were 1949); C, ethyl acetate-pyridine-water (10:4:3) (Jermyn suspended in cold distilled water (20 g. wet wt. in 100 ml. of & Isherwood, 1949). water). Walls were prepared as described by Armstrong The products were detected by the periodate-Schiff et al. (1960). The yield from a 9 1. culture was 0.8-1-4 g. reagents for a-glycols (Baddiley, Buchanan, Handschu- (Found: P, 2-87-3-3%). No whole cells were detected on macher & Prescott, 1956), molybdate reagent for phos- examination by electron microscopy. Samples (3 mg.) phoric esters (Hanes & Isherwood, 1949), aniline phthalate were hydrolysed for 3 hr. at 1000 with 2N-hydrochloric reagent for reducing sugars (Partridge, 1949), silver nitrate acid. After removal of hydrochloric acid by evaporation reagent for reducing sugars (Trevelyan, Proctor & Harrison, over potassium hydroxide, the residues were chromato- 1950) and ninhydrin reagent for amino acids (Consden & graphed in solvent A. The walls (204 mg.) were stirred for Gordon, 1948). 24 hr. in 10% trichloroacetic acid at 2°. After centrifuging Paper electrophoresis. Paper electrophoresis was carried the wall residues were suspended in trichloroacetic acid out on washed Whatman no. 4 paper in 0-05M-sodium and re-extracted. Ethanol (2 vol.) was added to the com- tetraborate at 5v/cm. for 16 hr. The glucosides were bined solutions and the resulting precipitate (42 mg.) located by spraying with 10% acetic acid in butanol, collected as before. This material was pure ribitol teichoic allowing to dry and then spraying with 1% sodium meta- acid. Acetone (2 vol.) was added to the supernatant from periodate solution containing 5% ofacetic acid and develop- this precipitation; the resulting precipitate (23 mg.) con- ing with the Schiff reagent in the usual manner. tained teichoic acid and polysaccharide. The wall residues Isolation of teichoic acid from Lactobacillus arabinosus (125 mg.) were freed from trichloroacetic acid by extraction cells. L. arabinosus 17-5 was grown in 9 1. batches for with ether. 16 hr. at 300 in the following medium: Oxoid Tryptone, In a similar extraction of walls (412 mg. Found: P, 20 g.; Oxoid yeast extract, 10 g.; sodium acetate, 10g.; 2-87%), 94 mg. of pure teichoic acid (Found: P, 6.5%), 126 A. R. ARCHIBALD, J. BADDILEY AND J. G. BUCHANAN 1961 22-4 mg. of teichoic acid-polysaccharide mixture (Found: were determined. The glucose value was corrected for P, 5 8%) and 200 mg. of extrac*ted wall residue (Found: destruction (5%) by acid during hydrolysis (Found: P, P, 07%) were obtained. In a ddition, the supernatant 6-44; glucose, 403; alanine, 12-1%. Ratio P:glucose: from the acetone precipitation contained 2-4 mg. of phos- alanine, 1:1-06:0.62). The reducing sugar present in an acid phorus, which corresponded to p,artially degraded or low- hydrolysate of teichoic acid was shown by assay with the molecular-weight teichoic acid. Glucostat reagent to be 98% of D-glucose. Examination of extracted walls. Extracted walls (3-7 mg.) Action of phosphodiesterase on teichoic acid. Teichoic acid were hydrolysed in 2N-hydroc*hloric acid (300,1A.) for (1-7 mg.), prepared from cell walls, was dissolved in water 24 hr. at 1000. After evaporation to dryness, the hydro- (3 0 ml.) and mixed with freeze-dried Crotalus atrox venom lysate was passed through colunans (0-5 ml.) of Dowex 50 (0*4 mg.) dissolved in 0- lM-glycine buffer, pH 9-0 (1 ml.), (H+ form) and Dowex 2 (CO. 2- foorm) resins. The eluate was and 1 mM-magnesium sulphate (0-3 ml.). The mixture was evaporated to dryness and examlined chromatographically then incubated at 370 for 24 hr. before being passed through (solvent A), together with unEextracted walls (1-3 mg.) a small column (0 5 ml.) of Dowex 50 (H+form) resin and which had been treated similarly. The amount (visual then adjusted to pH 5-5 with aqueous ammonia solution. estimation) of anhydroribitol pre,sent in the hydrolysate of The solution was freeze-dried, 0 2M-sodium acetate buffer, the extracted walls was less tha,n that present in the un- pH 5*5 (2 ml.), containing prostatic phosphatase (0-1 mg.), extracted walls containing ank equivalent amount of was added, and the volume was adjusted to 5 ml. with phosphorus. water. After incubation for 24 hr. at 370, the ratio in- Extracted walls (46 mg.) wer(e re-extracted by stirring organic: total phosphate was 0-145:1. After similar incu- with two successive portions (20 ml.) of 10% trichloro- bation with Crotalus adamanteus venom, the ratio was acetic acid at 700. Thewall residu4e(40 mg.), after removal of 0-093:1. A sample of teichoic acid which was incubated trichloroacetic acid by washinLg with ether, contained with prostatic phosphatase without previous treatmentwith 0-36% of phosphorus. No anhrydroribitol was detected snake venom gave after 48 hr. a ratio of 0 10: 1. On incu- when this residue (8.9 mg.) Mvas examined as above. bation with a purified calf intestinal phosphomonoesterase, Electron microscopy showed thabt the walls had not disin- the ratio was 0-145:1. tegrated appreciably. Further e-Ixtraction at 900 for 1 hr. Alkali hydrolysis and enzymic dephosphorylation. Teichoic with three successive portions off 10% trichloroacetic acid acid (980 mg.), isolated from whole cells by extraction at left a residue containing 0.04% of phosphorus. After this 20, was dissolved in N-sodium hydroxide solution (20 ml.) treatment the walls had disinttegrated completely to a and the resulting solution was heated at 1000 for 3hr. granular material. It appears thiat L. arabinosus walls con- A red-brown precipitate (probably ferric hydroxide) was tain small amounts of phosphorus other than that consti- removed by centrifuging. The combined supernatant and tuting teichoic acid. The nature of this phosphorus is not washings (50 ml.) were passed through a Dowex 50 (NH4+) known. resin column (30 ml.), which was then washed with water Teichoic Xwcid (200 ml.). The eluate and washings were concentrated to Acid hydrolysis. Teichoic aci(d (2-0 mg.) isolated from 10 ml. in vacuo or, in a second experiment, by freeze- walls was hydrolysed in 2N-hydlrochloric acid in a sealed drying. A sample (0.1 ml.) was removed and examined tube for 3 hr. at 1000. After eva6poration the samples were chromatographically in solvents A and B (see Table 1). examined by paper chromatograLphy in solvents A, B, and The pH of the eluate was adjusted to 5-5 with acetic C. Products are given in Table 1. acid, 0*2M-sodium acetate buffer, pH 5*5 (10 ml.), was Analysis. Teichoic acid (1.5 rng.), isolated from walls, added, and the solution was adjusted to 50 ml. with water; was hydrolysed in 2N-hydrochlorric acid (200 uL.) for 4 hr. at 4% of the phosphorus present was inorganic. Prostatic 1000. The hydrolysate was neut:ralized with ammonia and phosphatase (1-5 ml.) was added and the mixture was kept freeze-dried, and the residue was dissolved in water at 37°- Samples were removed for analysis after 24 hr. (5 0 ml.). Phosphorus (Chen, Toribara & Warner, 1956) (inorganic P, 69% of total P) and 48 hr. (inorganic P, 70% glucose (Park & Johnson, 1949) and alanine (Rosen, 1957' of total P). A further portion of phosphatase (1.0 ml.) was / added and incubation was continued for 24 hr. (inorganic P, 69% of total P). The volume was adjusted to 200 ml. Table 1. Products obtamzned by hydrolysis with water and the solution was passed through a column of teichoic acid of Dowex 50 (H+ form) resin (70 ml.) at 20. The filtrate, Teichoic acid was hydrolysed for 3 hr. in 2N-HCI or together with 250 ml. of column washings, was shaken with N-NaOH at 1000. The products3 were identified by chro- Dowex 2 (CO32- form) resin (20 ml.), and added to a matography. column of Dowex 2 (CO.2-) resin (20 ml.), which was then Acid Alkali washed with water (400 ml.). The eluate was evaporated hydrolysis hydrolysis under reduced pressure to small volume and freeze-dried. 1:4-Anhydroribitol + +* The resulting white solid (288 mg.) was examined chro- Ribitol + + * matographically in solvents A, B and C. Phosphates Glucose + - (520 mg.) were eluted from the Dowex-2 resin with N- Alanine + + sodium hydroxide solution (125 ml.) at 20. They were Glucosylribitol - + * isolated the eluate at 20 with Dowex 50 Diglucosylribitol by shaking (H+) Ribitol phosphates + +* (100 ml.), adding both the supernatant and resin to a Glucosylribitol phosphates + column of Dowex 50 (H+) resin (150 ml.) at 20, washing 1:4-Anhydroribitol 5-phosphate + - with water (11.), neutralizing with ammonia and freeze- Inorganic phosphate + drying. Those compounds listed in Tables 2 and 3 were * Trace. detected. Vol. 81 RIBITOL TEICHOIC ACID FROM L. ARABINOSUS 127 Examination of degradation products 1:4-Anhydroribitol. The eluate from the charcoal column (tubes 151-176) was evaporated to dryness in vacuo and the Fractionation of the neutral [product8. (a) On a cellulose anhydroribitol was purified by sublimation at 1450 and column. The neutral fraction (280 mg.) was dissolved in 104 mm. The resulting syrup crystallized after the addition solvent B (50 ml.) and applied to a Chromax paper column of a seed crystal of DL-anhydroribitol. Yield, about 1-5 mg. which had previously been washed with solvent. After of white crystals. The infrared spectrum was identical washing with three successive portions (10 ml.) of solvent, with that of authentic D-1:4-anhydroribitol. Polarimetric elution was commenced. Fractions (15 ml.) were collected, examination in a 1 dm. tube of a solution containing about and the elution of products was followed qualitatively by 0-5 mg. in 1-4 ml. of water gave a rotation of + 0.02-0.030. paper chromatography and quantitatively by micro- Bibitol. Chromatographically pure ribitol crystallized periodate oxidation (Dixon & Lipkin, 1954) on 0-1 ml. from ethanol as rosettes of white needles (23 mg.), m.p. samples of eluate after removal of solvent in vacuo. 990 (Found: C, 39-8; H, 8-2. Calc. for CrHlOj: C, 39-5; Most ofthe fractions contained mixtures, the composition H, 7.9 %). The infrared spectrum was identical with that of of which was determined by descending chromatography an authentic sample of ribitol. on Whatman 3mM paper with solvent B (see Table 2). (b) On a charcoal-Celite column. A sample of teichoic Identifiation of 4-O-(ac-D-glucopyranosyl)-D-ribitoI acid (500 mg.) was treated with alkali and prostatic phos- Chromatographically pure 4-glucosylribitol crystallized phomonoesterase as before, and the neutral fraction from with difficulty from aqueous ethanol as rosettes of white this was dissolved in water and passed through a column of needles (22 mg.), m.p. 125-1260, [u]22 + 106° in water (c, 1-2) equal parts of charcoal and Celite (total weight, 50 g.). The (Found: C, 40-0; H, 7-8. C11H22010,HH20 requires C, 39-8; elution of products was followed by paper chromatography, H, 7-2%. Ratio ribitol:glucose, found 1:0-94). It showed and appropriate fractions were combined and evaporated a band at 1631 cm.-' in the infrared, characteristic of under reduced pressure (Table 3). Mixtures were separated compounds containing water of crystallization. on Whatman 3mM paper. The 3 glucoside was obtained Acid hydrolysis. Samples ofthe glucoside (about 0-5 mg.) pure only after repeated chromatographic separation from were hydrolysed in 2N-hydrochloric acid (100pd.) for 3 hr. the 4-glucoside. After this purification all sugar deriva- and 24 hr. The hydrolysates were neutralized with am- tives were adsorbed on short charcoal-Celite columns and monia and examined by paper chromatography in solvent eluted with 15% ethanol. The solvent was removed under A. After 3 hr. the glucoside was completely hydrolysed to reduced pressure and the product finally freeze-dried. ribitol, anhydroribitol and glucose; after 24 hr. the pro- Unhydrolysed phosphates. The unhydrolysed phosphates ducts were anhydroribitol and glucose. consisted mainly of a monophosphate of diglucosyhribitol. Periodate oxidation and borohydride reduction. The glucosylribitol (1-0 mg.) was dissolved in 1% sodium meta- Table 2. Cellulose separation of products from the periodate solution (0-4 ml.) and the solution kept at room action of alkali and phosphatase on teichoic acid temperature for 24 hr. Sodium borohydride (2.0 mg.) was then added; after a further 24 hr. 2N-hydrochloric acid For details see text. (0-2 ml.) was added and the solution was heated at 1000 for Fraction no. Composition 30 min. After evaporation under reduced pressure the mixture was examined by ascending chromatography in 1-59 60-97 Anhydroribitol, glycerol ethyl acetate-pyridine-water (7:2:1) (Viscontini, Hoch & 98-145* Ribitol, 4-glucosylribitol, glucose (trace) Karrer, 1955). A single product was observed which 146-155 4-Glucosylribitol rapidly gave a purple colour with the periodate-Schiff 156-226 4-Glucosylribitol, 3-glucosylribitol reagents and had B. 0-45 (glycerol had R, 0-45, erythritol 227-300t Mono- and di-glucosylribitol had Rp 0-37 and ethylene glycol had RF 0.58). 301-460 Diglucosylribitol Quantitative periodate oxidation. Glucosylribitol (7.4 mg.) * Also contained a trace of a compound with ribsitol 1-19 was dissolved in water (10 ml.), 0-04m-sodium metaperio- (solvent B). date (5 ml.) was added, and the volume adjusted to 20 ml. t Also contained a trace of a compound with Rribitol 0-29 with water. A control experiment containing no glucosyl- (solvent B). ribitol was also performed. Solutions were kept in the Table 3. Charcoal chromatography of products from the action of alkali and phosphatase on teichoic acid For details see text. Fraction no. Eluent Composition 1-14 15-29 Glycerol 30-39 ' Water Glycerol, ribitol 40-83 Ribitol 84-130 131-150 Anhydroribitol (trace) 151-176 2 % Ethanol Anhydroribitol 177-200 Anhydroribitol (trace) 201-221 3 % Ethanol 3- and 4-Glucosylribitol 222-247 4% Ethanol 4-Glucosylribitol 248-320 6% Ethanol 3- and 4-Glucosylribitol, diglucosylribitol 321-390 765% Ethanol Diglucosylribitol 391-411 9-0% Ethanol Diglucosylribitol (trace) 128 A. R. ARCHIBALD, J. BADDILEY AND J. G. BUCHANAN 1961 dark at room temperature and samples were withdrawn at glyceric acid, prepared by enzymic dephosphorylation of intervals for determination of periodate (4 ml. samples), D-glyceric acid 3-phosphate, oxidized 2 mol.prop. of pyr- formic acid (5 ml. samples) and formaldehyde (0.4 ml. idine nucleotide, whereas DL-glyceric acid oxidized 1 mol.- samples) (Hough, Powell & Woods, 1956). The formic acid prop. of nucleotide at a reduced rate. was titrated with 0-01 N-sodium hydroxide solution to pH 6-4. After 24 hr. the glucosylribitol had consumed Identification of 3-0-(oc-D-glucopyranosyl)ribitoI 4-00 mol.prop. of periodate and 1-02 mol.prop. of formalde- Chromatographically purified 3-O-(a-D-glucopyranosyl)- hyde had been produced. After 48 hr. 4-10 mol.prop. of ribitol, from which all 4-glucosylribitol had been removed, periodate had been consumed and 1-06 mol.prop. of was adsorbed on a charcoal-Celite column (300 mg.) which formaldehyde and 1-43 mol.prop. of formic acid were was then washed with water, and the glycoside was eluted produced. On addition of an excess of sodium hydroxide with 10% ethanol. The eluate was evaporated to dryness solution and back-titration with 001N-oxalic acid, the under reduced pressure and the residue was dissolved in value obtained for formic acid was 1-90 mol.prop. water, then freeze-dried to a white powder (1-7 mg.). It Oxidation and hydrolysis to glyceric acid. The glucosyl- had [a]20 + 78±200 in water (c, 0-284) and a ratio glucose: ribitol (1-6 mg.) and o-methyl glucoside (2-0 mg.) were ribitol of 0-97:1. The amount of glucosylribitol in this separately dissolved in 0-15M-sodium metaperiodate solution, as estimated by determination of glucose in an (0 5 ml.), and the solutions were kept at room temperature acid hydrolysate, was 1-55 mg. for 48 hr. to ensure complete oxidation. The solutions were The solution was quantitatively transferred to a test passed through columns of Dowex 50 (H+ form) resin tube and the volume adjusted to 3-25 ml. with water; the (0.5 ml.) and Dowex 2 (acetate form) resin (0 5 ml.). The concentration of glucosylribitol in this solution, deter- columns were washed with water (10 ml.), the eluates were mined by estimation of the glucose produced on acid freeze-dried, and acetic acid was removed in vacuo. The hydrolysis, was 0-421 mg./ml. The remainder of the residues were dissolved in a solution (2 ml.), 100 ml. of solution was used for quantitative periodate oxidation which contained 417 mg. of benzoic acid and 565 mg. of studies. barium hydroxide. Bromine (0-02 ml.) was added to the Controlled oxidation with sodium metaperiodate. The ice-cold solutions which were then shaken in stoppered glucosylribitol solution (10 ml.) was mixed with 6 mm- tubes for 1 hr. and kept for 60 hr. at room temperature. sodium metaperiodate solution (2 ml.) and kept in the Air was passed through the solutions to remove excess of dark at room temperature. A control containing no glu- bromine and the volumes were reduced to 0-8-1-0 ml. by cosylribitol was also prepared. Samples (0-20 ml.) of each evaporation under reduced pressure. Benzoic acid was solution were diluted to 10 ml. with water and the perio- removed by centrifuging and washed with a little ice-cold date content was determined at intervals. Formaldehyde water. The volume of combined filtrate and washings was was determined in other samples (0-4 ml.). After 6 hr., reduced in vacuo to 0-5 ml., 5N-sulphuric acid (007 ml.) 4-1 mol.prop. of periodate were consumed, whereas after was added to each tube, and the solutions were heated at 24 hr. 4-8 mol.prop. of periodate were consumed and 100° for 2 hr. Barium carbonate (40 mg.) was added to 2-2 mol.prop. of formaldehyde were formed. After 72 hr., each and the mixtures were shaken at intervals during 5-1 mol.prop. ofperiodate were consumed and 2-3 mol.prop. 24 hr. Insoluble barium salts were removed by centrifuging of formaldehyde were formed. and washed with water. The supernatant andwashingswere Oxidation with sodium metaperiodate at pH 8-0. The passed through columns of Dowex 50 (NH4' form) resin glucosylribitol solution (0-35 ml.) was mixed with 0-1m- (0-5 ml.) and evaporated to dryness in vacuo. The glyceric phosphate buffer, pH 8-0 (1-70 ml.), 0-3m-sodium meta- acid was purified by chromatography in solvents A and B. periodate (0-27 ml.) and water (100 ml.). A control Paper strips containing glyceric acid, together with experiment containing no glucosylribitol was also prepared corresponding control areas of paper, were cut out and and both solutions were kept in the dark at 37°. Samples eluted with water or 0-01 N-ammonia solution. (0-4 ml.) were removed for determination of formaldehyde. The eluates were diluted to 2-0 ml. with water. Samples and after 24 hr. 3 03 were removed for chemical assay of the glyceric acid by After 12 hr., 3-12 mol.prop. mol.prop. chromotropic acid (Bartlett, 1959) or by oxidation with of formaldehyde were produced. 0-1 mM-sodium metaperiodate (Dixon & Lipkin, 1954). Identification of Oxidation was complete in 1 hr., 1 mole of periodate being consumed by each mole of glyceric acid. Samples (0-2- 3:4-di-(O-CX-D-glucopyranosyl)-D-ribitol 0-4 ml.) were then mixed with 3-5 ml. of a solution com- Diglucosylribitol was precipitated by cooling its solution posed of 0 01 M-phosphate buffer, pH 7-3 (12.0 ml.), in aqueous ethanol. It was a deliquescent, microcrystal- reduced diphosphopyridine nucleotide (about 4 0 mg.), line, white powder (30 mg.) which changed form at 1060 0-IM-magnesium sulphate (0-2 ml.), 0-2M-adenosine tri- and melted with decomposition at 118-121'. It had phosphate (0.2 ml.), rabbit-muscle enzyme (10 mg.) and [a]D +1370 in water (c, 1-08) (Found: C, 42-2; H, 7-1. yeast hexokinase (12 mg.). In a control experiment, water C17H320,5,6H20 requires C, 41-6; H, 6.8%. Glucose:ribitol (0-2-0-4 ml.) was mixed with 3-5 ml. ofthe enzyme solution. ratio, found 1-92:1). The hygroscopic nature of the material Oxidation of the pyridine nucleotide was followed prevented satisfactory measurement of its infrared spec- spectrophotometrically at 340 m,u, and under the above trum, but a strong absorption at 1639 cm.-', characteristic conditions was usually complete after 30 min. Oxidation of hydrated compounds, was observed. was calculated from the change in extinction at 340 m,z. Acid hydrolysis. Diglucosylribitol (about 0-5 mg.) was The glyceric acid from x-methyl glucoside oxidized hydrolysed in 01 N-hydrochloric acid (100!p.) for 90 min. 2-0 mol.prop. of pyridine nucleotide, whereas that from the The hydrolysate was neutralized with aqueous ammonia glucosylribitol oxidized 1-9 mol.prop. Authentic D- solution and examined chromatographically in solvent B. Vol. 81 RIBITOL TEICHOIC ACID FROM L. ARABINOSUS 129 Unhydrolysed diglucosylribitol (about 50%), glucose, and rupture of bacteria with a Mickle vibrator followed approximately equal parts of 3- and 4-glycosylribitol were by differential centrifuging, the technique being the only compounds detected. In 2N-hydrochloric acid adapted from that of Salton & Home (1951). This at 1000 for 4 hr. the products were glucose, ribitol and method was anhydroribitol. the most satisfactory one for pre- Periodate oxidation and borohydride reduction. Acid paring cell walls from L. arabinosus for the present hydrolysis of the reduced product of periodate oxidation purpose. Much of the teichoic acid could be gave two glycols; the principal one had Rp 0 45 and the extracted from walls by treatment with cold 10 % minor one had RF 0-39 (glycerol had R, 0 45 and erythritol trichloroacetic acid at 20 for several hours. Ex- had R. 0.37). tracti6n at 370, or even higher temperatures, was Quantitative periodate oxidation. Diglucosylribitol more complete but these vigorous conditions led to (4.99 mg.) was dissolved in a little water, 0 02M-sodium the presence of contaminants in the final product. metaperiodate solution (5 ml.) was added and the volume The ease of extraction of this teichoic acid is com- was adjusted to 25 ml. with water. The solution, and a parable with that for Bacillus subtilis (Armstrong control solution, were kept in the dark at room tempera- ture and samples were removed for analysis. After 36 hr., et al. 1960), and is consistent with the view that 5-10 mol.prop. of periodate were consumed and 1-80 mol.- most, or possibly all, of the teichoic acid in prop. of formic acid were formed; after 72 hr., 5-21 mol.- bacterial walls is held in the wall structure by prop. of periodate were consumed and 1-98 mol.prop. of ionic linkages. formic acid and 1-08 mol.prop. of formaldehyde were L. arabinosu walls contain about 3 % of phos- formed. The oxidation product was stable at pH 6-9, but phorus, 90 % of which can be accounted for as was unstable above pH 7, giving rise to acidic products. ribitol teichoic acid after extraction with trichloro- Partial acid hydrolysis. The diglucoside (10 mg.) was acetic acid solution. The nature of the small dissolved in Ol N-hydrochloric acid and heated at 1000 for amount of unextracted phosphorus is unknown. 90 min. The solution was neutralized with ammonia solution and ions were removed by use of a charcoal-Celite This organic phosphate is not a teichoic acid. column. The resulting monoglucosylribitols were purified Similar amounts of unextracted organic phosphates by chromatography in solvent B, adsorption on charcoal have been observed in the walls of other bacteria and elution with ethanol as before, and identified as follows: studied in this laboratory. (a) 4-O-(X-D-GluCopyranosyl)-D-ribitol. This compound, Pure teichoic acid was obtained from the extract obtained by hydrolysis of the diglucoside, was indistin- by precipitation with ethanol, dissolving in water guishable on paper chromatography (solvent B) and colour and reprecipitation with ethanol. It contained reactions from authentic 4-glucosylribitol. On acid 6-44 % of phosphorus and gave on acid hydrolysis hydrolysis, glucose, ribitol and anhydroribitol were the alanine, glucose, inorganic phosphate and the only products detected. The glucose: ribitol ratio was of ribitol 0-92: 1. The pure glucosylribitol (0-8 mg.) was oxidized with known acid-degradation products phos- periodate, the product was reduced with borohydride then phate (Armstrong et al. 1958b). The ratio phos- hydrolysed, and fragments were examined as before. phorus:glucose:alanine was 1: 1-06:0-62. The Glycerol (RF 0.45) was the only product detected with the amount of alanine depended upon the conditions periodate-Schiff reagents. A sample (1.2 mg.) of the of preparation but the molar proportion was glucosylribitol was oxidized with sodium metaperiodate always less than 1. At present it is assumed that followed by bromine, then hydrolysed and the resulting a molar proportion of about 1 occurs in teichoic glyceric acid was examined as before. With x-methyl acid within the wall and that partial loss of alanine glucoside as a reference compound, the enzymic procedure takes place during isolation. showed that 90% of the glyceric acid formed had the D- configuration. A sample of alanine from this teichoic acid was (b) 3-0-(ox-D-Glucopyranosyl)ribitol. This compound readily oxidized by kidney D-amino acid oxidase from the partial acid hydrolysis gave no reaction with (experiment by Dr F. C. Neuhaus) and thus has the aniline phthalate and had the same chromatographic D-configuration, a feature common to all teichoic properties in solvent B as had 3-0-(O-D-glucopyranosyl)- acids examined in this Laboratory. Moreover, the ribitol. It also gave the characteristic yellow colour with alanine residues react readily at room temperature the periodate-Schiff reagents. On acid hydrolysis, glucose, with dilute ammonia or hydroxylamine, giving ribitol and anhydroribitol were the only products detected. alanine amide and hydroxamate respectively. These The glucose:ribitol ratio was 0-97:1. On oxidation with observations, and the presence of a typical ester sodium metaperiodate at pH 8-0, 2-80 mol.prop. of form- at 1751 cm.-' aldehyde were produced. carbonyl absorption band in the infrared, indicate that the alanine residues are joined through their carboxyl groups to hydroxyl DISCUSSION groups of either the glucose or ribitol residues. It Analysis of this teichoic acid and paper-chro- has been shown that the alanine residues in the matographic characterization of hydrolysis pro- ribitol teichoic acid from B. subtilis are joined to ducts were carried out on material which had been ribitol and not to glucose (Armstrong et al. 1961). prepared from isolated walls as well as from whole The general properties of this polymer and the cells. The walls were obtained by mechanical nature of the products of its acid hydrolysis are 9 Bioch. 1961, 81 130 A. R. ARCHIBALD, J. BADDILEY AND J. G. BUCHANAN 1961 consistent with a structure in which ribitol ant isomer of glucosylribitol also crystallized as a residues are joined together through phosphodiester monohydrate. It had the same R, as the glucosyl- linkages. This similarity to the ribitol teichoic acid ribitol from B. subtilia teichoic acid in two solvents from B. subtilis walls is in contrast with the nature and behaved similarly on electrophoresis, but and number of glucosyl residues attached to ribitol. differed slightly in the colour given with the When the phosphodiester linkages in the B. subtili8 periodate-Schiff reagents. The blue colour given by polymer were hydrolysed by alkali, a mixture of the ,B-glucoside from B. subtilis closely resembles phosphomonesters was produced almost quanti- that from ,8-methyl glucoside, whereas that shown tatively. Removal of phosphate from the esters by the glucoside from L. arabinosus possesses a through the action of prostatic phosphatase gave blue-grey component. cx-Methyl glucoside gives a a high yield of 4-0-($-D-glucopyranosyl)-D-ribitol, marked blue-grey colour in this test. and it was concluded that the teichoic acid is a Acid hydrolysis of the glucosylribitol gave polymer in which each glucose residue is attached glucose, ribitol and anhydroribitol, whereas under in a regular manner to each ribitol in the chain. more vigorous conditions equimolar amounts of When a similar procedure was applied to the glucose and anhydroribitol were formed. These teichoic acid from L. arabinosus, a mixture of were separated from each other by paper chro- products was obtained. In order to obtain suffi- matography. Anhydroribitol was determined by cient of these for adequate characterization, larger- oxidation with periodate, and glucose by reaction scale experiments were carried out on hydrolysates with ferricyanide (Park & Johnson, 1949). The of teichoic acid prepared from extracts of whole, high positive rotation ([oc]"? + 1060) of this glucosyl- defatted bacteria. Such preparations were less ribitol suggested that it is an oc-glucopyranoside. pure than those from walls, and contained hydro- This is consistent with the observation that it was lysis products from the intracellular glycerol not hydrolysed with the f-glucosidase preparation teichoic acid that is known to be present in this which readily hydrolysed the 4-0-(fl-D-glucopyran- organism (cf. Baddiley & Davison, 1961). osyl)-D-ribitol from B. subtilis, and is also con- The main products of hydrolysis were a mono- sistent with the earlier conclusion that the high glucosylribitol and a diglucosylribitol, and appreci- rotation of the teichoic acid from L. arabinosus is able amounts of ribitol were also obtained from due to the presence of ax-glucosyl residues. There is preparations made from some batches of organisms. thus a marked difference between the teichoic acid In addition, small amounts of 1:4-anhydroribitol from L. arabinosuw and that from B. subtilis in and an isomeric monoglucosylribitol were detected which all glucose linkages possess the f-configura- in the hydrolysate. The anhydroribitol is probably tion. not a structural unit in the teichoic acid, but could The position of attachment of glucose to ribitol arise during the alkali treatment. A trace of 1:4- was established by oxidation with sodium meta- anhydroribitol is formed by the prolonged action of periodate; 4 mol.prop. of periodate were consumed hot alkali on ribitol 5-phosphate (unpublished and 2 mol.prop. of formic acid and 1 mol.prop. of observation by Mr D. A. Applegarth), and it is formaldehyde were produced. For complete possible that phosphodiesters in a teichoic acid liberation of formic acid it was necessary to add an would yield the anhydro-compound more readily excess of sodium hydroxide. The 4-0-(fl-D-gluco- than would the simple phosphomonoester. Occa- pyranosyl)-D-ribitol from B. subtilts teichoic acid sionally a small amount of an isomeric diglucosyl- behaved similarly, and this is believed to arise ribitol has been detected in hydrolysates, but this from the intermediate formation of a glucosyl-D- is not a regular product. erythrose, which then gives a formyl ester of the The hydrolysis products were separated from final oxidation product. The course of the oxida- each other by chromatography on charcoal, tion has been discussed fully (Armstrong et al. followed in some cases by preparative paper 1960). It follows from the analysis that the glucose chromatography. Anhydroribitol was isolated must be attached to a hydroxyl at position 2 or 4 crystalline by sublimation. It had the same chro- in the ribitol residue (cf. I and II). This conclusion matographic mobility in several solvent systems is supported by the observation that the trialde- as authentic 1:4-anhydroribitol and its infrared hyde formed by oxidation of the glucoside with spectrum was identical with that of optically active periodate, on reduction with borohydride and acid 1:4-anhydroribitol, which differs from the spectrum hydrolysis, gave glycerol but not ethylene glycol. of the racemate. Insufficient material was avail- The two possible structures (I and II) for the able for accurate measurement ofits optical activity, glucosylribitol can be distinguished from each but the solution was slightly dextrorotatory, sug- other by considering the stereochemistry of their gesting that the sample was the D-isomer. Ribitol oxidation products. was isolated crystalline and identified by direct The structure (I), on oxidation with periodate, comparison with synthetic material. The predomin- then with bromine, would give the tricarboxylic Vol. 81 RIBITOL TEICHOIC ACID FROM L. ARABINOSUS 131 acid (III); this would yield on acid hydrolysis tion. The enzymic method for determining D- 2 mol.prop. of D-glyceric acid (IX). On the other glyceric acid 3-phosphate is similar to that used hand, the altemative structure (II) would give the previously for establishing the configuration of the tricarboxylic acid (VI), which would yield on acid ribitol phosphate residue in cytidine diphosphate hydrolysis 2 mol.prop. of DL-glyceric acid. In a ribitol, and is known to be specific for the D-series similar degradation of the glucosylribitol from B. (Baddiley, Buchanan & Carss, 1957a). 8Ubtil8 teichoic acid, the configuration of the When the glucosylribitol from L. arabinosu8 glyceric acid was determined from its optical teichoic acid was subjected to this procedure, the rotation in the presence of molybdate, but in the resulting glyceric acid, after phosphorylation, present work an altemative procedure was adopted. oxidized 1-9 mol.prop. of pyridine nucleotide: The glyceric acid was isolated from the reaction authentic D-glyceric acid, and the glyceric acid mixture by paper chromatography, eluted from the obtained from a-methyl glucoside, oxidized 2 mol.- paper and then determined chemically by oxida- prop. of pyridine nucleotide; DL-glyceric acid tion of a sample with sodium metaperiodate. Its oxidized 1 mol.prop. of nucleotide. Thus, the configuration was established by phosphorylation glyceric acid from the glucosylribitol must have the with adenosine triphosphate in the presence of D-configuration and the glucosylribitol is correctly crude yeast hexokinase to give D-glyceric acid 3- represented as 4-0-(OC-D-glucopyranosyl)-D-ribitol phosphate (V). This was determined by its ability (I). to oxidize reduced diphosphopyridine nucleotide in This combined chemical and enzymic method for the presence of a rabbit-muscle-enzyme prepara- determining the structure (I) of the glucosylribitol CH'-OH ,-OH

OHHO/ E

OH HO 0 EO1H2C |CH2*OH

(I) (II) (1) IO4 I(1) IO4- I (2) Br2 (2) Br2

vH,*OI1 HE-OH H0 \0 H02 0 HOXCHO HO H2C HO2C ( HO2C 0CH2OH HO-H2C CO2H (III) (VI) |H+ H+

C2H Kinase DL-Glyceric acid OH CH2-OH (V) (IV) H,O-POH2 9-2 132 A. R. ARCHIBALD, J. BADDILEY AND J. G. BUCHANAN 1961 is conveniently carried out on a small scale ribitol. Confirmation of the structure 3-0-(oc-D- (3A4jumoles of glycoside), and could be applied to glucopyranosyl)ribitol (VII) was obtained by the determination of the stereochemical series of oxidation at pH 8 with a large excess of sodium many other glycosides. It has been used success- metaperiodate at 37°. Hough & Perry (1956) have fully here for determining the stereochemical shown that under these conditions compounds series of the glucose in ac-methyl D-glucoside and, that would be expected to be oxidized to deriva- with minor modification, has been applied in other tives of malondialdehyde are readily susceptible to work on the structure of nucleotides containing a further degradation known as 'over-oxidation'. D-glucose or D-mannose (Blumsom & Baddiley, In a glucosylribitol this could only occur if the 1961). It is likely that this procedure will have glucosyl residue is at the 3-position. When the considerable general use in the carbohydrate glucoside was oxidized in this way, 3 mol.prop. of field. formaldehyde was produced, showing that 'over- After several chromatographic separations, a oxidation' had occurred and therefore that the pure specimen (1.7 mg.) of an isomeric glucosyl- glucose was attached to the hydroxyl at the 3- ribitol was obtained from a teichoic acid hydro- position of the ribitol. lysate. This was non-reducing and on acid hydro- This structure was supported by the character- lysis gave glucose, ribitol and anhydroribitol. istic yellow colour given by the compound on Equimolar amounts of glucose and anhydroribitol paper chromatograms sprayed with the periodate- were formed on vigorous acid treatment. The Schiff reagents. This behaviour has been observed optical rotation of the glucosylribitol ([aC]22 + 78+ previously with 3-substituted ribitol derivatives 200) suggests that the glucosyl linkage is a, and this (Baddiley, Buchanan & Carss, 1957b). is supported by the stability of this glucoside in the The diglucosylribitol was precipitated from presence of a fl-glucosidase. aqueous ethanol as a white deliquescent powder, On oxidation of the isomeric glucosylribitol with which was probably microcrystalline. Vigorous sodium metaperiodate in dilute solution in the acid hydrolysis gave glucose and anhydroribitol in dark at room temperature, 4-5 mol.prop. of perio- the molar ratio 2: 1. The high positive optical date were consumed and 2 mol.prop. of formalde- rotation indicated that both glucose units are hyde were produced. It follows that the glucose is present as oc-glucosyl residues; this conclusion was attached to the hydroxyl at position 3 on the supported by the failure of a ,B-glucosidase to hydrolyse the glycoside. The diglucosylribitol consumed 5 mol.prop. of CH2-OH periodate, giving 2 mol.prop. of formic acid and 0\~~ 1 mol.prop. of formaldehyde. Reduction of the oxidation products with borohydride, then acid HO hydrolysis, gave glycerol and erythritol. Careful acid hydrolysis of the diglucoside gave glucose and HO OH two isomeric non-reducing monoglucosides of ribi- HO.'H2C tol. It follows that the two glucose residues are CH2OH each directly attached to the ribitol and cannot be (VII) present in a glucosylglucose unit.

(VIII)

(IX) Vol. 81 RIBITOL TEICHOIC ACID FROM L. ARABINOSUS 133 From the above evidence it follows that the two phosphate from the products with a phosphatase, glucose residues in the diglucoside must occupy gave mainly a mono- and a di-glucosylribitol. positions 3 and either 4 (2) or 5 (1) of the ribitol. Small amounts of anhydroribitol and an isomeric The 3:4- and 3:5-diglucosyl-D-ribitol structures are monoglucosylribitol are also formed, and occasion- shown in (VIII) and (IX) respectively. ally traces of an isomeric diglucosylribitol have The isomeric structures were distinguished from been detected. Teichoic acid preparations from each other by studies on the monoglucosides ob- some batches of micro-organisms but not others tained by careful acid hydrolysis. The two mono- yield appreciable amounts of ribitol. glucosides were separated from each other by 3. The principal monoglucosylribitol has the paper chromatography, and both gave equimolar structure 4-0-(OC-D-glucopyranosyl)-D-ribitol (I). amounts of glucose and anhydroribitol on vigorous This has been established by studies on optical treatment with acid. One was identified as 3-0- rotation, hydrolysis, periodate oxidation and the (xC-D-glucospyranosyl)ribitol (VII) by paper-chro- stereochemistry of degradation products. matographic comparison with the 3-glucosylribitol 4. A general method is described for determining isolated previously. It also gave the characteristic the stereochemical series of sugars from their glyco- yellow colour with the periodate-Schiff reagents, sides on a microscale. This depends upon oxidation and on periodate oxidation at pH 8, 'over-oxida- of the glycoside with periodate then bromine, tion' occurred, 3 mol.prop. of formaldehyde being hydrolysing the product with acid, and determining formed. the configuration of the resulting glyceric acid by The other glucosylribitol from the diglucoside enzymic phosphorylation then oxidation of re- was indistinguishable on paper chromatograms duced diphosphopyridine nucleotide in the pre- and in colour reactions from 4-0-(X-D-gluco- sence of a rabbit-muscle preparation. pyranosyl)-D-ribitol (I). On oxidation with perio- 5. The monoglucosylribitol formed in small date, followed by reduction with borohydride and amounts is 3-0-(c-D-glucopyranosyl)ribitol (VII). acid hydrolysis, it gave glycerol but neither The structure of this compound was established by erythritol nor ethylene glycol. The 5(1)-glucosyl- methods similar to those adopted for the isomeric ribitol which would be formed by partial hydrolysis 4-glucoside. of structure (IX) would give ethylene glycol by this 6. The diglucoside is 3:4-di-0-(oc-D-glucopyrano- procedure; thus, structure (IX) is untenable for the syl)-D-ribitol (VIII). On careful acid hydrolysis it diglucoside. Moreover, the aldehyde obtained by yields glucose and a mixture of 3- and 4-glucosyl- oxidizing the diglucoside with periodate is particu- ribitol identical with those isolated before. larly labile towards alkali. This can be explained 7. The teichoic acid from the walls of Lacto- readily on the basis of a 3:4 (2) structure as in bacillus arabinosus differs markedly from the other (VIII). ribitol teichoic acid which has been studied in The structure (VIII) was distinguished from the detail, i.e. that from Bacillus subtilis, in that both isomeric 2:3-di-0-(oc-D-glucopyranosyl)-D-ribitol by mono- and di-glucoside residues are present and the stereochemical analysis of the 4(2)-glucosylribitol glycosidic linkages all have the a-configuration. obtained by partial hydrolysis of the diglucoside. We thank the Medical Research Council, the Nuffield The glyceric acid obtained by oxidizing the glu- Foundation and the Rockefeller Foundation for financial cosylribitol with periodate then bromine followed support. by hydrolysis had the D-configuration. Conse- REFERENCES quently, the glucosylribitol must be the 4-glucoside (I), and the diglucosylribitol must be correctly Armstrong, J. J., Baddiley, J. & Buchanan, J. G. (1960). represented as 3:4-di-O-(oc-D-glucopyranosyl)-D- Biochem. J. 76, 610. Armstrong, J. J., Baddiley, J. & Buchanan, J. G. (1961). ribitol (VIII). Biochem. J. 80, 254. Although the glucosylribitol (I) and the diglu- Armstrong, J. J., Baddiley, J., Buchanan, J. G. & Carss, B. cosylribitol (VIII) are the principal components of (1958a). Nature, Lond., 181, 1692. the teichoic acid from L. arabino8us walls, their Armstrong, J. J., Baddiley, J., Buchanan, J. G., Carss, B. relative amounts and the relative amounts of & Greenberg, G. R. (1958b). J. chem. Soc. p. 4344. ribitol and isomeric glycosides vary in different Baddiley, J., Buchanan, J. G. & Carss, B. (1957a). J. chem. samples of teichoic acid. Soc. p. 1869. Baddiley, J., Buchanan, J. G. & Carss, B. (1957 b). J. chem. Soc. p. 4058. STMMARY Baddiley, J., Buchanan, J. G. & Carss, B. (1958). Biochim. biophy8. Acta, 27, 220. 1. It is confirmed that the teichoic acid from the Baddiley, J., Buchanan, J. G. & Greenberg, G. R. 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Biochem. J. (1961) 81, 134 Optical Rotation of Soluble Feather Keratin

BY N. E. DWELTZ* Department of Phy8ic8, Univer8ity of Madra8, Madra8 25, India AND V. MAHIADEVAN Department of Phy8ical Chemi8try, Univer8ity of Madras, Madras 25, India (Received 6 February 1961) A new structure has recently been proposed for and the extraction made in a 50 ml. mixture of lOM-urea, feather keratin by Ramachandran & Dweltz (1961), 0 1 M-NaHSO, and 0 05M-sodium phosphate buffer at a pH which is based on a collagen-like triple helix and of about 8-5 for 48hr. at 600. The extract was cooled to room which is in good agreement with its detailed X-ray temperature and filtered, and the clear protein solution collected. This was then made up to 50 ml. with the urea diffraction pattern. In support of this, they mixture and its optical rotation measured accurately at showed that feather keratin and collagen exhibit 250 with a Hilger standard polarimeter. remarkable resemblances in their infrared-absorp- The protein concentration of feather keratin in solution tion spectra and birefringence, in addition to the was determined from the non-diffusible N content. For nature of oriented crystallization of inorganic salts this purpose 4 ml. portions of the urea-bisulphite-protein on the two fibrous proteins and in their behaviour solution were pipetted into cellophan sacs and dialysed on treatment with chemical reagents. In the against running tap water for various periods ranging from present paper details are given of the measurement 1 to 7 days in steps of 1 day each. After the fourth day the of the optical rotation of soluble feather keratin, estimated N was constant, but decreasing very slowly, probably because a fraction of the protein content was also which again is found to be close to that of collagen diffusing out of the cellophan sac. A correction for this was both in sign and magnitude. made from the rate of change of concentration observed from the fourth to the seventh day. MATERIALS The estimation of the N content in each case was carried AND METHODS out as follows: The contents of each dialysis sac was White goose-feather keratin was used in these experi- quantitatively transferred after dialysis to a 50 ml. flask ments. The samples were first thoroughly degreased by and digested with conc. H2SO4 (A.R.) for 3 hr., over a washing in a number of changes of benzene, then cleaned microburner. Reaction was completed by adding 1-2 ml. by washing in a number of changes of distilled water and of 30 % H202 so as to remove any faint-yellow colour subsequently air-dried. produced during digestion. The digest was then cooled in an The solution of soluble SH-keratin of feather was ice bath and neutralized with NaOH (A.R.) added in drops, prepared according to the method of Woodin (1954, 1956). to a pH of approx. 7, and then made up to 100 ml. The N A weighed amount (about 1 g.) of dry feathers was taken in this neutralized digest was estimated by nesslerization. The colorimetric measurements were made in a Klett- * Present address: ATIRA, Ahmedabad-9, India. Summerson photoelectric colorimeter by using a no. 54