STORAGE PRODUCTS IN CAULERPA B45 some properties of soluble 1 agreen alga Coulerpa simpliciuseula. Plant 159-68, KN. 1850, ‘The structure of the mi in Porphyra umbiticantis, J. Chem, Soe 8. Loh, GA. 1975. Cytological and Chemica the Wound Response in Calera protijera University of South Florida, Tampa, (Diwertation Abst 6, 2020), 9. Love, J. Machie, W.. MeKinnell, J. W. x Peveival, E. 163, Slarch-type polysucchatides isolated from the green seqweeds, Enleronmorphe compressa, Ula lactuca, Cla dophara rupestris, Codinn fragile aml Chaetomorpha ‘enpiliaris. J Chien, Soe. YWGESIT7-¥2. 10, Mackie, LM. & Percival, E1960, P J. Phiycor. 8, 315-348 (1977) ALC 1. ACID AND SUL the green weed! Cailerpu filiformis, Past H. & glucan of e amylopectin type. f. Chem. Sor, 1900:288) U1, Stith. MoM. & Stone, BLAS 1978. Chemical ¢ of the ceil walls of Lolium mudofilosin etwtosper 12 Southworth, D. &. Dickinson, D. 1975, jel syuthase from Lilian’ longiflorwm pollen. Plant Physiol 5688-7. Swank, ROT. & Munkies, KD. 197L Molecular wel slssis of oligopeptices by electrophoresis in polyacrsha Inide gel with sodium dodeeyl sulphate. vel. Biochem. S477 TH. Trench, RK. Trench, Mt 1 Syibiotic chloroplasts: their photosynthetic products aid onteibuition to mucus synthesis in (80 maine sugs. fol Bell, 12333549, MAN BLUE: A QUANTITATIVE AQUEOUS ASSAY FOR FATED POL SACCHARIDES! Je Ramus Alcian Blue, a cationic copper phthalocyanine dye complexes with the anionic carboxyl and halfester sulfaie groups of acidic algal polysaccharide in aqueous solution to form an insoluble precipitate The quantity of dye removed from solution is. pro- portional to the quantity of polyanion in solution, ‘and this principle forms the basis for the quantitative determination of acid and/or sulfated algal pobysac- charides. The assay is linear between 0 and 100 ng/ml agar, alginic acid, carrageenan, pectin and Porphyridium aerugineum Geit. pobysaecharide. In addition, the technique is used to determine the anion density of acid polysaccharides on a molar or weight equivalency basis, Key index words: agar: Alcian Blue; alginic acid; anionic polyacchavides; assay, algal polysaccharides; carrageenan; pectin; polysaccharides, acid: polysne chavides, sulfated: Porphyridium; quantitative assay Phe procedures most used for the qua analysis of acid sullated polysaccharides are strong mineral acid-chromogen type assays, specifi cally the phenobsulluric aeid (1) and anthronesul- lurie acid (12) assays. Both are colorimetric: con centrated sulfuric acid hydrolyzes glycosidic li titative ages, + accepted: 30 May 1977 1 thology, Yale Univesity, New Haven, Comnectient 06520 nil phenol or anthrone react with the monosac charide components to produce a colored product Both assays measure only total carbohydrate and de stroy the biopolymer in the process but neither mea. sures anion density AL present, polysaccharides localized on the sur faces of cukaryotic algae (6) are the subject of i sive research. ‘The polysaccharides may be important cell surface antigens, compose the cell wall or capsule and, it some instances, have gelling properties uselul in commercial processes antl products. Most are acidic (anionic) polymers, bearing cither carboxyl (GOO ) oF hallester sullate (-OSO, ) reactive groups x both. The names of the polysaccharides are i¢ and include such heteropolymers as agar, n, furcellaran, porphyran and ginie acid, carrageen Polyanions will electrostatieally complex with cu ions and the resulting complexes often precipitate For example, the acidic polymer produced by the uni: cellular red alga Porphyridinm acruginenm Geit precipitates. when complexed with the detergents cetyl pyridinium chloride or cetyl trimethyl am. monium bromide or the cationic dye Meian Blue (8) Alcian Blue (AB) has been used for histochemical ization of acid and sulfated biopolymers in both 10) and algal (7) tissues. It iy a soluble form nil derives. its catio346 J. RAMUS TaBue 1. Anion densities for acidic polysaccharides based on quantitative Atcian Blue (AB) binding iopalrmer Special Heatnont 1 Agar nome 08200 37200 0.100 Alginie Acit SUA 23.2°1.9 0.60.05 Carrageen; none 07201 82505 0.10¢01 Peet one Loot L605 O42 Porphyridinm L202 97209 0 acrugineun none 08 refused in 0366 methanolic HCL 0000) 0.0200 0.6000 100C,0.1M NaOH 2303 107514 OSR=08 100 €.0.19 Na.CO, 3 02-07 ase Na* (as Ch) 02008 (3M Na* (as Cl) OM Car? (as Cr) ASRS 42 Seieh end Booty. ‘Aon sitessugar residue properties from isothiowronium groups. When a constant amount of heparin, a mucopolysaccharide from mammalian tissues, is mixed with increasing amounts of Alcian Blue and the amount of AB com- bined assesed by measuring the dye left in the supernatant, the amount precipitated remains. re- markably constant (11). Seowt et al. (11) first suggested that AB could be used in quantitative assay for polyanions. In the following, 1 demonstrate can be used in quantitative analysis of acid and sul: fated polysaccharides, provided the anion density from batch to batch is’ constant. Alternatively, the assay can be used to measure anion density on a molar or weight equivalency basi has definite value to those seeking an alternative that avoids biopolymer destruction. hat Alan Blu MATERIALS AND ARETHODS Alcian Blue isthe quarierary methylp-tcluene sulphonate ot copper teta-L:pyridyl phthaloeyanin. Alcan Blue AGN (In grain blue 1; C1. 71240; Matheson, Coleman and Bell) was dis oie in 05 a actic ack pH 25, to a concentration of 10 gyal To el tal pico miple es ided Beal 05. i Xolie acd and the solution ete; then Tmt of the AB Solution adel, and the solution again mixed "The solution fas allowed to sand several hours, mixed, then centrifuged a0 AM dissed in 01 4 acetic acid abuas maximum fig 610 nm ith halémaximum absorption etween 373 and. 700 him. "Therefos absorption was read at 610 hm, andthe differ tice between the blank andthe sample taken ax proportion torte poljunion cinoertrnien. A eshien convaining 100 9 ‘NB/mt‘03 a acetic acil gives an absorption of a. 10. The Srelght in ug of AB complexe by the simple cn be calculate from the difference abworption between blank and sample mmltipied by the concentration of AB to give om ‘heol-sulfuric acid asays for comparative meantrements of polyaccharides used the method of Duboin ta (0) Porplyridium aeruginewm (UTEX. 73) wa grow i Tiga cule @) and niynctharide pret tata from the cell-free supernatant the prewnce of O01 8 and 3 vol ethanol. The polysiechatide was washed in absolute ethanol, dried in yaco over CaCl, then ground 1 a fine powler. When appropriate, the polysaccharide was ¢ sulfated by refluxing in 0.3% methanolic-HCI according to the method of Haq and Percival (0), om explants of the sascukar plant Arabidopsis thaliana (L-) Heynh, were grown in gyrorotary-shaken axenic liquid culture ‘on the “Linsmaier-Skoog” median (5). ‘The cells in suspension were separated from the medium by centrifugation at 10.000 4, and the clear supernatant solution used in test procedures. Anionie polysiecharides were supplied. by the manufactasers as dried powders, and incladed! agar (Bacto-Agar, Difeo Laboratories), crrrageenan (Ca-salt HMR, Marine Colloids, Ine Rockland, Maine), alginic acid (Na-salt, Type IV, Sigma Chem Go) “and pectin (apple, Mann” Research Laboratories, Mountainview Ave, Orange, New York). Once dried, acidic ppolysiccharides do not completely redisperse in hot aqueous solution. "Therefore, they were stirred at 100 C for Th and centrifuged upon cooling at 27.000 g. The resul natant as assayed by the phe stock soluti Two neutral plant polysiceharides were used as dye-binding, specificity control substances: agarose (Agarose A43, L'tnda Biologique Francaise, 33, Qual da Moulin de Cige—2-Gen- nnevillies, France) and starch (Goluble potato, Fisher Scientifi) The hexuronic acid q-D-galacturonic acid (Mann Research Laboratories) was used 8 «control for polymer precipitation. ali pectate and from Porphyridiwn aeruginewm were determined with AB (Tuble 1). Primary data were taken from aqueous solutions of heatcispersed. polymer at concentrations ‘of 10, 20, 40, 70 and 100 gg/ml, as determined by the phenol sulfuric acid method, Mean saliies ~ the range of variation are sgiven for the concentrations tested. For calculations resulting in the number of anionic sites per sugar residue, the molecular ‘weight of Aleian Blue SGN was 1840 daltons (8) and the sugar residues was 140 daltons ke Both Porphyridiwn aerugineum and stem explants of Arabidopsis thaliana when grown in liquid suspen- sion cultures released acid and/or sulfated polysac- charides into the medium, ‘The culture supernatants of both organisms contained Alcian Blue binding material and the complexes removed the dye from solution in a linear fashion proportional to the amount of centrifuged culture supernatant added to the assay mixture (Fig. 1). ‘These binding materials were anionic biopolymers in their native state, i. uunalfected by chemical recovery and_redispersion Fife tadidon to nacive polyyacchalides che granclar or powdered anionic polysaccharides in aqueous so- lution were assayed for AB binding and polymer con centration by the phenolsulfuric acid assay. ‘These were agar and carrageenan (sulfated galactans pro- duced by red algae), alginate (polyuronide produced by kelps) lar plants). All gave linear assays to concentrations, of 100 yg polymer/ml (Fig. 2), equivalent in sensitiv: ity to the anthrone and phenol-sullurie acid assays To ensure complete binding, the assay mix was al- owed to stand several hours at 25 C before centrifu Noninear results were obtained when theALCIAN BLUE POLYSACCHARIDE ASSAY 37 500) Arabidopsis w $ 6 ug AB Complexed 100] o 1 2 4 6 8 ml Culture Supernatant tative removal of Aleian Blue (AB) from solu tion by native exereed polyanion trom liquid cultures of Porphyridiunt aeruginewm and Arabidopsis thaliana: cach data point yepresents mean value for 3 simples, assay mix was immediately centrifuged. “The contol polysaccharide (stareh, agarose, galacturonic acid) did not remove AB from solution (Fig, 2), In addition to giving a linear, reproducible, quan- titative assay for alginic acid, ag, ageenain pectin and Porphyridivm polysaecharide, the AB technique can be used to compute the anion density of acid and or sullated polymers (i. to compute the nu sites/sugar residue). It is assumed that each anion site binds a single molecule of AB, al though there may be up to four positively charged (isothiouronium) groups/molecule. This opens the possibility that adjacent anions might share an AB molecule or that electrostatic bridges might form between separate polymers through AB. The assump- pn appears valid for several reasons: first, the amount of dye bound per unit polymer is consistent over a tenfold concentration range of polymer (Table 1): second, the theoretical limit for alginic ‘acid, whose uronic acid content approaches 100%, is one molecule AB complexed /uronic acid residue (ce Table 1, column 111). ‘The disparity may result from impurities in the sample, decarboxylation or methy! tion during the manufacturer's isolation process, the purity of the Alcan Blue SGN, or an error in the estimation of the molecular weight. Nevertheless, the maximum theoretical amount of dye which alginic acid could bind is ca. 34 x 10% mol /yg polymer and, the computed value is 23 X 10! mol/ng polymer (Table 1). ‘The anion density of agar, carrageenan and pectin is more variable. AIL are heteropolymers. whose anion density depends on the species source of the isolation and the process itself 300} 3 200) Te) : é ad a 2 100 vests te ———earrageenen| ug Polysaccharide Fic. 2 Assay of hot water dispersed acid andl neutral polysaccharides by quantitative precipitation of Alcian Blue (AB) from aqueous solution: each data point represents mean Nalue for 3 samples ‘The acid polysaccharide produced by P. aerugineum is up to 7.6% by weight halbester sulfate and carries, a small quantity of uronide (8). Computed anion density is approximately one for every four sugar residues (Table 1). Theoretical calculations give a maximum halfester sulfate content of ca. 12% by weight and that number is reduced by the presence of uronidl The chemical alteration of Porphyridium polysac- de has predictable effects on AB binding. Desul- on and methylation by refluxing in 0.3% metha- nolie-HCI reduced binding to zero (Table 1). Boiling, the polysaccharide in weak alkali had no effect, indicating that the halfester sulfates are stable. ‘The auldition to the assay mix of monovalent cation (sodium) to 0.2 M1 and divalent cation (calcium) 10 0.1 xt had no effect on binding, indicating the high binding affinity of AB for polyanions. “However higher concentrat educed the quantity of dye bi ‘The utility of the AB technique in the determina: tion of production rates of acidic polysaccharides is, obvious. However, the technique allows the addi tional advantage of qualititative analysis of produced polymer. The culture supernatant polymer of P. Gerugineun was assayed through lig, log and station- 'y phase for anion density. “The theoretical maxi mum number of AB binding sites is ea, 33. 10!/ug, polymer. The cells produced a uniform anion density polymer of 10 x 10%/ng polymer with little signili- cant variation during growth phase progression (Fig. »)‘M8 |. RAMUS is & stationary > 8 AB Molecules Complexed Culture Age (days) Fic. 3. Acidie groups (per unit weight) of acid potyse haride excreted by Porphyridium aerugineum into liquid eu ture plotted as a function of culture age (molecules AB > 10" ount!/ug polyanion): each data point represents mean for 8 culties, vertical bars repre e of values Most colorimetric assays give the least amount of coloration with the least amount of assayed substance, thereby being very senisitive at low sample concentra- tions. ‘The AB assay relies on a difference measure ment of chromogen remaining in solution after complex formation and precipitation of polymer- chromogen complex. It is east sensitive at low substrate concentrations. remedy for this was by dissolving the washed biopolymer-AB complex in electrolyte solutions and measuring colorimetrically the dye released, Quantitative solution proved iffi- cult, especially for sulfated polymers. This is consis- tent with the observation that half-ester sulfates have an exceedingly high critical electrolyte concentration (CEC) (lor carrageenan C 25 M Na’, 20.1 Mg’) and the value for carboxyl groups is 5-10 times lower (CEG = 0.38 xt Na’, 0.15 wt Mg’ for alginate; 0.18 mt Na*, 0.02 | Mg for pectin) whereas the value for phosphate groups is very low (11). De- possible shortcoming, the AB assay is equal in sensitivity and reproducibility to the strong min eral acid-chromogen assays. Carbocyanine, another organic ¢ onic dye, is used in colorimetric estimation of acidic polysaccharides (2). Binding of the dye to polyanion results in a shift in the dye absorption maximum to a longer wave- length, ie. a metachromatic shift. ‘The magnitude of the spectral shift is polymer specific. Although the assay is sensitive (0.5-5 jg), it has at least three dis advantages: i) reagent solutions are very unstable ii) reagent solutions are very light labile; and, iii) light absorption is very salt concentration. sensitive. The AB assay sulfers none of these disadvantages. work was supported by National Science Foundation Re hy Grant BMS. 7500436. 1 wish to thank James R. Wong ‘suspension cultures of Arabidopsis thaliana stem. ex ubois, M. Gilles, K. A., Hamilton, J. D., Rebers, Po AL & Smith, F. 1956. Colorimetric method for determination ‘of sugars and related substances, nal. Chen. 28:350-6. 2, Edstrom, RD. 1969, A colorimetric method for the de termination of mucopolysaccharides and other acidic polymers. Anal, Biochem, 29:421- E. 1960. Encyclopedia Lid. Lon 4. Hag. 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