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Biochem. J. 51, 70.Bray, H. G., Humphris, B. G., Thorpe, W. V., White, K. &Wood, P. B. (1952b). Biochem. J. 52, 416.

Brodie, B. B., Axelrod, J., Shore, P. A. & Udenfriend, S.(1954). J. biol. Chem. 208, 741.

Burns, J. J., Evans, C. & Trousof, N. (1957). J. biol. Chem.227, 785.

Calvin, M., Heidelberger, C., Reid, J. C., Tolbert, B. H. &Yankwich, P. F. (1949). 18otopic Carbon. New York:John Wiley and Sons Inc.

Clayson, D. B. (1953). Brit. J. Cancer. 7, 460.Fewster, M. E. & Hall, D. A. (1951). Nature, Lond., 168,78.Gessner, T. & Smith, J. N. (1960). Biochem. J. 75, 172.Henrich, F. (1921). Ber. dtech. chem. Ges. 54, 2507.Henrich, F. & Wagner, B. (1902). Ber. dtsch. chem. Ge8. 35,

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Science, 128, 120.Ishidate, M., Hogiya, S., Hashimoto, Y. & Takitani, S.

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p. 13. London: Chapman and Hall Ltd.

Biochem. J. (1960) 77, 503

The Chemical Composition and Structure of theCell Wall of Hydrodictyon africanum Yaman

BY D. H. NORTHCOTE, K. J. GOULDINGDepartment of Biochemi8try, University of Cambridge

AND R. W. HORNECavendish Laboratory, University of Cambridge

(Received 21 January 1960)

The coenobium of the green alga Hydrodictyonafricanum consists in a single net of about 500 cellsor coenocytes of similar size and stage of develop-ment. The net-like colonies can easily be grown inculture, the individual cells reaching a size of 2 mm.or more in diameter. The organism thus givesmaterial which is very well suited to a study of

plant cell growth and in particular to the develop-ment of the cell wall. The walls are large and can beobtained free from the cell contents by a variety oftechniques including microdissection, which is bothmechanically and chemically very gentle. The workreported here is a microscopical and chemical studyof the cell walls prepared from fully developed cells.

D. H. NORTHCOTE, K. J. GOULDING AND R. W. HORNE

The species H. africanum has been described byYamanouchi (1913), Wigglesworth (1928) andPocock (1937). Hydrodictyon reticulatum has beengrown under laboratory conditions and used inphotosynthesis and other studies (Neeb, 1952;Schon, 1955; Pirson & Schon, 1957; Pirson, 1957).

EXPERIMENTAL

Material used and general analytical methodsOrganism. Hydrodictyon africanum Yaman. (Culture no.

236/2) was originally obtained from the culture collection ofalgae and protozoa in the Botany School, Cambridge, andwas kindly given by Mr E. A. George.The nets were grown in a sterile soil extract prepared in

1 lb. Kilner jars (500 ml.) without the metal cap or rubberring. The soil (approx. 50 g.), which was collected from thegardens near the laboratory, was mixed with CaCO3 (approx.0.4 g.) and covered with 420 ml. of tap water and steamedfor 1 hr. It was allowed to cool to room temperature andsteamed again for a further hour. The medium was used inthe jars, as prepared, without removal of the soil whichsettled after about a week. The jars were stored for about a

month before use when the liquid above the soil was a clearlight amber colour and contained 0-13 g. of solid matter/l.For the germination of zygospores a similar medium was

prepared but the soil used was Kettering loam.The zygospores were kept in media in which the nets had

developed and had produced gametes. After the collapse ofthe net the zygospores could be seen as an orange layer on

top of the soil: in this form they could be stored for at least3 years. The spores were germinated by transferring themto a fresh medium kept at 18 20° in a light intensity of 600lux for periods of 16 hr. followed by 8 hr. of darkness. Thelight was supplied by two 40w, 2 ft. natural fluorescenttubes and two 40w tungsten filament lamps. After 3-4weeks small green nets approx. 1-2 mm. in diameter couldbe seen and when these were transferred, one net to a jar,and grown under the same conditions as that used forgermination, they developed into large nets 5-10 cm.

across in which the individual cells had a diameter of1-3 mm. Growth to this size usually took about 4 weeks.Each net normally consisted of about 250 or 500 cells

(approx. 28 or 29) and some colonies with up to 1000 cells

were obtained.Isolation of the cell-wall material by mechanical breakage of

the cells. The nets were removed from the medium andwashed in distilled water and the individual cells separatedby gently pushing the cells apart with a small spatula.Generally any one cell was round and taut and joined tofour other cells, two at one pole and two at the other (P1. 1,fig. 2 inset). The cell was broken with a hypodermic needleand the contents were washed out by injecting distilledwater. The process was watched under a microscope ( x 12-5)and was continued until no cell contents could be seen

adhering to the wall. The wall appeared as a transparentcolourless membrane which was transferred with fineforceps to a beaker ofwater and washed by gentle agitation.The damp walls were freeze-dried.

All the analyses were carried out on material dried at0-01 mm. Hg over P205 at room temperature. Total N was

determined by micro-Kjedahl digestion (Chibnall, Rees &Williams, 1943) followed by distillation and titration.

Total P was determined according to Fiske & Subbarow(1925).Chromatography and elettrophoresis of sugar8. Descend-

ing chromatograms were run on Whatman no. 1 papers withpyridine-ethyl acetate-water (1: 2: 2, by vol.) for 14 hr. andethyl acetate-acetic acid-water (3:1:3, by vol.) for 24 hr.(Jermyn & Isherwood, 1949). Electrophoreses were run at210v in an apparatus similar to that described by Consden& Stanier (1952) with borate buffer, pH 9-2. The sugar spotswere coloured in all procedures with aniline hydrogenphthalate (Partridge, 1949).

Chromatography ofamino acid8. Two-dimensional chroma-tograms were run on Whatman no. 52 paper with butanol-acetic acid-water (4:1:5, by vol.) and phenol-m-cresol (1: 1,v/v) buffered at pH 9-3 with borate according to Levy &Chung (1953). The spots were coloured with a 0.3% soln. ofninhydrin in butanol.

Chemical -investigation of the cell wall

The average dry weight of one cell was 26Hg.(833 cells, wt. 21 9 mg.) and the cell wall was 10 2 jg.(616 walls, wt. 63 mg.), 39.2 %. The cytoplasm ofthecell could be seen as a thin layer ofgranular materialclosely applied to the cell wall [P1. 1, figs. 2 (inset)and 5] and inside this, occupying the bulk ofthe cellvolume, was the large vacuole.

Elementary analysis and mineral content. The totalN was 0 65% and the total P 0.168% of the dryweight. When maintained at red heat (approx. 8000)in a platinum boat in a stream of clean dry air for1 hr., 17-23 mg. of walls yielded a white ash;0 05 mg. (0 3%).

Lipid analysis. The lipid was determined byboiling the cell walls (57 3 mg.) with aq. 80% (v/v)methanol (20 ml.) for 2-5 hr. and centrifuging andsiphoning off the alcoholic supernatant from thewalls, which were redried at 0 01 mm. Hg overP205. The material was re-extracted with boilingdry ether (20 ml.) for 2-5 hr. Yield of extracted cellwalls was 53-2 mg. and the wt. loss of the walls was4-1 mg. (7.2 %). The methanol and ether extractswere combined and evaporated under reducedpressure to a thick yellow oil (3-8 mg.).Acid hydrolysis. (i) For monosaccharide con-

8tituents. The whole cell walls (5 mg.) were sus-pended in 0 1 ml. of 72% (w/v) H2SO4 and left atroom temperature for 24 hr. when most of thematerial dissolved. The solution was diluted withwater (0 7 ml.) and heated at 950 for 6 hr. in asealed tube, then neutralized with BaCO3, filtered,and the filtrate was evaporated to dryness at 00.Galacturonic acid, glucose, mannose, xylose and avery slight trace of arabinose were found in thehydrolysate by chromatographic and electro-phoretic investigations.

(ii) For amino acids. Whole cell walls (7 mg.) werehydrolysed by 6N-HCI (3 ml.) at 1050 for 24 hr. in asealed tube. The hydrolysate was evaporated at 250undervacuum, dissolved in water and re-evaporated

504 1960

EXPLANATION OF PLATES 1-3

In all Figs.: o, outer edge of wall; i, inner edge of wall; 1, laminations; p, pores;pm, pore membrane; m, microfibrils.

PLATE 1

Figs. 2, 3. Whole cell wall stained with light green. The circular pads at the poles of the cell can be seen.

Fig. 2 (inset). Portion of a whole colony showing the arrangement of the cells.

Fig. 4. Portion of cell wall treated with 0 5% NaOH at 4° for 24 hr. Shadowed with Pd/Au at an angle of60°. The microfibrillar organization can be seen.

Fig. 5. Transverse section of a whole small cell (1 mm. in diameter) fixed in osmic acid. The three layers ofthe wall are visible as a thin outer and inner membrane of dark staining material and a thick middle layerhardly stained.

PLATE 2. All the sections are cut transverse, see Fig. 1 (p. 506).

Fig. 6. Wall treated with 3% NaOH for 24 hr. and stained with osmic and phosphotungstic acids. A pore canbe seen passing obliquely across the wall. The dark thin outer layer shows very clearly and in the middle layerthere are indications of the thin laminations running parallel to the cell surface.

Fig. 7. Untreated wall fixed with osmic acid. The membrane of the pore passing straight across the section appearsto be continuous with the inner and outer layers of the wall.

Fig. 8. Wall treated with 3% NaOH for 24 hr. and fixed in osmic acid. A detailed view of a pore at the innersurface of the wall is shown.

Fig. 9. Untreated wall fixed with osmic acid. A branched pore is visible.

PLATE 3

Fig. 10. Section of untreated wall fixed in osmic acid and cut tangentially (see Fig. 1). Fine microfibrils(30-40A in diameter) can be seen as closely packed structures running parallel to the edge of the section.A lamination runs across the section at right angles to the microfibrils. The pores can be seen as holes bounded bythin membranes.

Fig. 10 (inset). Microfibrillar structure at higher magnification.

Fig. 11. Untreated wall fixed in potassium permanganate. The fine laminations of the middle layer can be clearlyseen. The outer edge of the wall is formed by the darkly stained material and the inner layer appears granulated.

(Facing p. 504)

I'he Bitoch^eibiccl .Joioriad, -VXol. 77, ..-o. :3 Plate I

il _ l cmi

FF'' ' ' s | ,-:" ..~~~~~~~~~~~~~~~~~~~~~~~I

Fig. 2 Fig. 3

Fig. 4 Fig. 5

D. H. NORTHCOTE, K. J. GOULDING ANI) R. IV. HORNE

The Biochemical Journal, Vol. 77, Xo. 3 Plate 2

'QE

m

PM

Fia. 6 Fig. 7

... ....... ...

PM

V

vo....... ...

Fig. 8 Fio'. 9

D. H. NORTHCOTE, K. J. GOUJLDING AN DRI. W. HORNE

The Biochemical Journal, Vol. 77, No. 3 Plate 3

Fig. 10

Fig. 11

D. H. NORTHCOTE, K. J. GOULDING AND R. W. HORNE

CELL WALL OF HYDRODICTYON

several times. Aspartic acid, glutamic acid, serine,glycine, alanine, tyrosine, valine, leucine/isoleucine,lysine and smaller amounts ot threonine, phenyl-alanine, proline, hydroxyproline, arginine andcysteine were identified in the hydrolysate.

Poly8aocharide8 of the ceU wall. Walls (53.2 mg.)from. which lipid had been extracted were treatedwith 24% (w/v) KOH (10 ml.) at room temperatureunder N2 for 4 hr. The residue from the extractionwas well washed with water by suspension andcentrifuging. Six washings were required to give asupernatant with the same pH value as the waterused for washing. The washings and alkaline extractwere combined and the residual a-cellulose fractionwas dried over P205 at 0 01 mm. Hg. Yield of whitetranslucent material was 39-4 mg. (68.7 %). It stillretained the shape of the original cell wall.The alkaline solution of the hemicellulose frac-

tion was neutralized at 40 with cooled acetic acidand evaporated under reduced pressure to half itsvolume. It was poured into a volume of ethanol sothat the final concentration of alcohol was 85 %.A precipitate did not appear immediately but after2 hr. a discrete gelatinous precipitate formed andsettled. It was left for 24 hr. when it was centri-fuged, washed with ethanol and dried. Yield ofslightly brown coloured material was 9-0 mg.(15.7 %).The a-cellulose fraction (6 mg.) was hydrolysed

with 72% (w/v) H2SO0 according to the method ofSaeman, Moore, Mitchell & Millett (1954), and achromatogram of the neutralized hydrolysateshowed glucose and mannose only. These sugarswere estimated in the mixture by the method ofWilson (1959) and the molar ratio was found to be1:1. Even preparing an ac-cellulose fraction fromthe walls with 24% (w/v) KOH+4% (w/v) boricacid (Jones & Painter, 1957), or by boiling with10% (w/v) NaOH in the absence of air for 4 hr., didnot appreciably alter the ratio ofglucose to mannosein the preparation.The hemicellulose fraction when hydrolysed with

2N-H,S04 at 1000 for 6 hr. and neutralized with anion-exchange resin (Amberlite IR-4B, CO.2- form)contained glucose, mannose with trace amounts ofxylose and very slight traces of arabinose. Theglucose and mannose were the main sugars in thehydrolysate and these were present in the molarratio of 2:3 respectively. Galacturonic acid couldbe detected in the material when the hydrolysis wascarried out with 2N-H2SO4 or 3% HNO3 and thesolution neutralized with BaCO3. The hemicellulosesolution did not colour with I2 solution and con-tained no starch.

This hemicellulose fraction was investigated byzone electrophoresis on glass paper with boratebuffer, pH 9-2 (Fuller & Northcote, 1956). Onespot was observed at a distance of 3 5 cm. from the

origin towards the cathode, 2:3: 6-tri-O-methyl-D-glucose moved 13 cm. and starch used as a markergave a spot at 6-4 cm.

Microscopical examination of the cell wallThe cell walls were examined by the optical and

electron microscopes. The cell walls were seen ascolourless transparent bags which were easilystained by methylene blue, zinc iodide chloride andlight green (Pl. 1, figs. 2, 3). After the cell had beenemptied of its contents four circular thin pads couldbe seen situated in pairs at opposite poles on theoutside of the walls and corresponding to the posi-tions where the cell had been in contact with itsneighbours in the intact colony (P1. 1, figs. 2, 3).These pads were about 110 p in diameter for a cellapprox. 2 mm. in diameter (six cells measured, sizerange 110-111 ,) and in optical section seemed to beslightly raised at the edges (P1. 1, fig. 2).A comprehensive examination of the cell wall was

made under the electron microscope with a SiemensElmiskop operating at a high-tension voltage of80 kv and also with a Phillips E.M. 100 operating ata high-tension voltage of 60 kv. The preparationswere supported on nitrocellulose and 'Formvar'(polyvinyl formal) films stabilized with evaporatedcarbon, which were mounted on copper grids (New200, diam. 2-3 and 3 mm.; V. A. Howe and Co. Ltd.,London, W. 11).The whole wall was too large to be directly

observed with the electron microscope and wasbroken into small pieces by suspension in water in asmall blender (3 ml.) driven at 14 000 rev./min. bya top drive (M.S.E. Ltd., London, S.W. 1) for10 min. The pieces of wall were centrifuged, resus-pended in water and allowed to dry at room tem-perature in air on to the specimen grids. These wereshadowed with Pd/Au, by using standard shadow-casting techniques. The shadowing angle was 600.

Sectioned material was prepared by fixing thewall in buffered (pH 7-3) osmium tetroxide solution(1 %, w/v) (Palade, 1952) or KMnO. (0.6 %, w/v)(Luft, 1956). In some instances the preparationsstained with osmium tetroxide were further stainedby leaving them for 24 hr. in a solution of phospho-tungstic acid (1 %, w/v) in 70% ethanol during thedehydration procedure (Huxley, 1957). The speci-mens afterwashing and dehydration were embeddedin a mixture of 85% (v/v) of butyl methacrylateand 15% (v/v) methyl methacrylate which waspolymerized with 1.5% (w/v) of benzoyl peroxideat 500. The sections were cut with glass knives in amicrotome described by Porter & Blum (1953).

Unmectioned material. Microfibrils could not beseen in untreated preparations of the cell wall butwere apparent when the wall fragments were treatedwith 0.5% (w/v) NaOH at 40 for 24hr. (P1. 1,fig. 4). The microfibrils were closely packed and in

Vol. 77 505

D. H. NORTHCOTE, K. J. GOULDING AND R. W. HORNE

any fragment examined the majority ran in onedirection but there were always some whichappeared to be oriented across this preferred direc-tion. They could not be obtained free from otherwall material.

Sectioned material. Both untreated cell walls andthose which had been soaked for 24 hr. with 3%NaOH at room temperature were used. There wereno obvious differences in the two preparations(Pls. 1 and 2, figs. 5-9). The sections of the wholecell and the cell wall (Pls. 1 and 2, figs. 5-9) stainedwith osmium tetroxide showed that the cell wallconsisted of three main layers, a thin outer darkstaining area approx. 250 A thick, a middle section5 IL across which hardly stained, and an inner layerof darker staining material which varied in thick-ness between approx. 200 and 1000A (P1. 2, figs.6-9). In the sections stained with osmium tetroxidethere were some indications of laminations (approx.250k thick) parallel to the outer and inner surfacesof the wall in the thick middle section (P1. 2, fig. 6)and these became very apparent when the materialwas stained with permanganate (P1. 3, fig. 11) .Occasionally sections were seen in which thelaniinations ran across the section at right angles tothose more commonly observed (P1. 3, fig. 10).

Al

Fig. 1. Diagram of a portion ofa cell wall containing a pore.Plane AEDC represents a tangential section and plane AEBa transverse section. The lines CFI and GHJ indicatelaminations in the wall. The three layers of the wall are notrepresented.

From other evidence, which is discussed below andwhich relates to pores seen in the wall, there isreason to believe that these latter sections weretangential. In Fig. 1 plane ABE represents atransverse section where the laminations runparallel to the inside and outside of the wall, andplane AEDC represents a tangential section inwhich the laminations (lines CFI and GHJ) appearacross the section more or less at right angles tothose in plane ABE. The sections cut in planeAEDC showed very detailed microfibrillar struc-tures closely packed and all running in one directionaround the wall. The diameter of the microfibrils inthesestainedpreparationswas 30-40k (P1. 3, fig. 10).

In most sections of the wall dark-staining stripsof tissue (diameter 0-7 ,u) bounded by a thin mem-brane 200k thick, could be seen passing across thewall from the inside to the outside of the cell(P1. 2, figs. 7 and 9). Sometimes they appeared torun very obliquely (P1. 2, fig. 6) and some werebranched (P1. 2, fig. 9), but more usually they wereunbranched and passed fairly straight across thewall. The membrane bounding the strip could beseen in some cases to be continuous with the layersat the inside and outside of the wall (P1. 2, figs. 7-9)and this suggested that the structures were porescontaining material which had a strong affinity forosmium tetroxide or that their dark appearancewas due to the obvious membrane structure aroundthem which took up the osmium very strongly. Theidea that these structures were pores was furtherstrengthened by the appearance of holes in thesections cut in the plane AEDC (Fig. 1). These canbe seen in P1. 3, fig. 10, to be oval (diameters1 Iu x 0-4 u) and bounded by a thin strongly stainingmembrane. The microfibrillar structure can be seento bend around these pores like the graining ofwood around a knot hole.

DISCUSSION

The cell wall of this alga is a large structurerepresenting over 39% (w/w) of the dry weight ofthe cell and measuring approximately 5 5 ,u thick.It is composed largely of ac-cellulose-like material(69%) which retains the shape and structure of theoriginal wall and is organized as a microfibrillarphase. The microfibrils are very fine (about 30-40kin diameter) and are closely packed and run in adefinite direction around the wall. However theac-cellulose-like material occurs as layers which liebetween thin laminations running parallel with thecell surface, and the direction of the microfibrils,although seemingly constant in any one layer, maybe different in the different layers. The size of themicrofibrils in this green alga is similar to that ofChlorella pyrenoido8a (Northcote, Goulding &Horne, 1958). Generally in higher plants the

506 1960

Vol. 77 CELL WALL OF HYDRODICTYON 507microfibrils are larger, ranging from about 50 to250i in diameter (Northcote, 1958).The lipid (7 %) which has a greater affinity for

osmium than the rest of the material appears to besituated as thin layers both at the inside and outsideof the wall and also as membranes for pores whichtraverse it.The hemicellulose material (16 %) and protein

(4 %, calculated from the nitrogen content) can beremoved from the wall without noticeably affectingits structure, and they therefore probably consti-tute the matrix material around the microfibrilsand might also be concentrated to form the thinlaminations which divide the x-cellulose-likematerial. On hydrolysis the hemicellulose materialgives glucose and mannose with small amounts ofxylose and arabinose which indicate that theprincipal polysaccharide present is a glucomannan.The oc-cellulose fraction also contains mannose in anamount equal to that of the glucose present. In thechemistry of wood the occurrence of these non-glucose sugars in oc-cellulose preparations is wellknown (Bertrand, 1899; Norman, 1937; Northcote,1958; Polglase, 1955; Wise, 1958). Recently, how-ever, the mannose polymers have been progressivelyremoved from the preparations by means of variousalkaline solutions (Hamilton & Quimby, 1957) andmixtures of alkali and boric acid (Jones & Painter,1957); and Rapson & Morbey (1959) claim to haveobtained a highly pure cellulose from wood, that isan a-cellulose preparation comparatively un-degraded but almost free of non-glucose sugars. Onthe other hand, Cronshaw, Myers & Preston (1958)have reported that an a-cellulose preparationobtained in small yield from the whole cells of amarine alga, Porphyra, contained mannose only. Itis, however, very difficult to detect small amountsof one sugar in the presence of large amounts ofothers. In addition the presence of non-glucosesugars in a-cellulose material can be explained intwo ways: (a) as contaminants from the hemi-cellulose of the continuous matrix surrounding andadhering to fibrils, or (b) as part of the polymer(s)forming the microfibrillar structure (Northcote,1958). The very close packing of the microfibrils inHydrodictyon and the difficulty of obtaining amicrofibrillar preparation, which when examinedby the electron microscope appeared free frommatrix material, makes an explanation for theoccurrence ofmannose in this ac-cellulose impossibleat present.

Galacturonic acid is present in the hydrolysatesof the whole wall, and this is probably derived frompectic substances. In the higher plant thesematerials are found in the middle lamella betweenadjacent cells, and in Hydrodictyon it might well bepresent in the thin circular pads which occurbetween neighbouring cells in the colony and which

can be clearly seen when the walls have been freedfrom cell contents.The pores bounded by a membrane are the most

obvious feature in the sections, and since the wall isa very large structure in this plant their function ismost probably that of transport. However theycannot be open holes from the outside to thevacuole since the osmotic pressure of the cell is suchthat when pricked it usually bursts. For a similarreason there can be no direct open connexion be-tween the cells of a colony, for they can easily beseparated without any obvious change.

SUMMARY

1. Cell-wall preparations of the coenocytes ofHydrodictyon africanum have been obtained. Thewalls are approximately 5-5 ,u thick and represent39-2% of the dry weight of the whole cell.

2. An analysis of the wall has been made withrespect to the following fractions: ox-cellulose frac-tion (69 %), hemicellulose fraction (16%), protein(4 %), lipid (7 %) and ash (0 3 %), which accountsfor over 96% of the material.

3. The cell wall appears as a transparent colour-less membrane and two pairs of thin circular pads,approximately 110Lu in diameter, can be seen atopposite poles corresponding to the position ofneighbouring cells in the intact colony. The wallsstain with the normal histochemical tests forcellulose.

4. The wall is made up of three layers, thin innerand outer layers and a thick middle layer. Themiddle layer is a two-phase system in which micro-fibrils approximately 30-40A in diameter are closelypacked and enclosed in a continuous matrix. Thislayer is further divided by very thin lamellae whichrun parallel to the cell surface, and the microfibrilsin any one section of it lie mainly in one directionaround the wall.

5. The hemicellulose fraction contains glucose,mannose and traces of xylose and arabinose. It isprobably present as a glucomannan. The oa-cellulosefraction is composed ofpolymers containing glucoseand mannose. The occurrence of the mannose in theoc-cellulose fraction with respect to the compositionof the microfibril is discussed.

6. Pores seen as oval in shape (1 I, x 0- 4,u indiameter) which are bounded by a dark-stainingmembrane traverse the wall.

REFERENCES

Bertrand, G. (1899). C.R. Acad. Sci., Paris, 129, 1025.Chibnall, A. C., Rees, M. W. & Williams, E. F. (1943).

Biochem. J. 37, 354.Consden, R. & Stanier, W. M. (1952). Nature, Lond., 170,

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The Determination of Amino Sugars in the Presence of AminoAcids and Glucose

BY C. CESSI AND FRANCA PILIEGOI8tituto di Patologia Generale, Univer8itc, Bologna, Italy

(Received 25 April 1960)

The presence of other sugars and amino acidsinterferes with the method for quantitative estima-tion ofglucosamine and galactosamine described byElson & Morgan (1933). The procedure, which isbased on the reaction of amino sugars with acetyl-acetone in an alkaline medium and subsequentdevelopment of a red colour by chromogens formedwith p-dimethylaminobenzaldehyde, has beenmodified by several authors. Many of these modifi-cations are quoted by Rondle & Morgan (1955) intheir paper dealing with the improvement of theestimation. Immers & Vasseur (1952) have exa-mined the interference arising from mixtures ofamino acids together with glucose under variousconditions. Schloss (1951) proposed a methodwhich overcomes such interference by takingadvantage of the fact that colour due to aminoacids and glucose fades after a comparatively shorttime. In his study of the mechanism of the reactionSchloss (1951) stated that a number of chromogenicproducts are formed by heating hexosamines withacetylacetone in alkaline media; partial fractiona-tion of the reaction mixture yielded one volatileliquid and two or more non-volatile solid chromo-gens. The use of a homogeneous fraction giving acolour with p-dimethylaminobenzaldehyde would

seem safer than a complex mixture for an analyticalprocedure. One of us (Cessi, 1952) developed amethod based on the separation of the volatilechromogen(s) for the estimation of amino sugars instrongly coloured solutions. The procedure has beennow adapted to the quantitative estimation ofamino sugars in the presence of amino acids andglucose.

EXPERIMENTAL

Materials and methods

Amino sugars. D-Glucosamine hydrochloride was ob-tained from Hofmann-La Roche and Co. (Basle, Switzer-land) and recrystallized from water by addition of conc.hydrochloric acid. D-Galactosamine was a kind gift fromProfessor W. T. J. Morgan. Aqueous solutions were madefrom hydrochlorides, and weights are referred to free sugars.

Acetylacetone reagent. Colourless, redistilled acetylacetone(b.p. 138-140°) was stored in the refrigerator and dissolved(1 ml.) in 100 ml. of 0 5N-sodium carbonate-sodiumbicarbonate buffer containing O-IM-sodium chloride.A buffer ofpH 9-8, as adopted in the proposed method, wasobtained by dissolving 23-02 g. of sodium carbonate, 2-76 g.of sodium bicarbonate and 5-84 g. of sodium chloride/l. ofsolution. The pH was checked after the addition of acetyl-acetone with a glass electrode standardized against 0-05M-sodium tetraborate and adjusted if necessary. The reagent