ANALYTICAL BIOCHEMISTRY93, 295--298 (1979)

A Partial Modification of the Carbazole Method of Bitter and Muir for Quantitation of Hexuronic Acids MASASHI KOSAKAI AND ZENSAKU YOSIZAWA

Department of Biochemistry, Tohoku University School of Medicine, 2-1, Seiryo-machi, Sendai 980, Japan Received June 13, 1978 The carbazole method of T. Bitter and H. M. Muir (1962, Anal. Biochem. 4, 330-334) for quantitation of hexuronic acids was modified partially. In this modification (procedure g), 0.2 M anhydrous sodium tetraborate was used instead of 0.025 M sodium tetraborate decahydrate. The color intensity of D-glucuronolactone was slightly diminished by this modification, however, that of L-iduronolactone significantly increased. Thus, the difference in color intensity between o-glucuronolactone and L-iduronolactone became very small. When evaluated by procedure g, the hexuronic acid content of dermatan sulfate was comparable to that of chondroitin sulfate A.

The carbazole reaction of Bitter and Muir (1) has been used widely for quantitation of hexuronic acids, because of its reproducibility, stability of color, specificity, and inexpensiveness of the reagents. In this method, however, the extinction coefficients of the color given by L-iduronic acid and L-iduronic acid-containing substances differed considerably from those given by D-glucuronic acid and D-glucuronic acidcontaining substances (1,2). In the previous papers (3,4), we reported that this difference could be made smaller in the presence of 0.1-0.2 M borate (sodium tetraborate decahydrate), so that the deamination products of heparin could be quantitated more accurately (3). This paper reports a further modification of the carbazole method of Bitter and Muir for more accurate quantitation of hexuronic acids in dermatan sulfate-type substances.

MATERIALS AND METHODS Materials. D-Glucurono-y-lactone was purchased from Tokyo-Kasei Company, Tokyo. 1,2 - Isopropylidine - fl - L - ido furanurono - y - lactone was a gift from Dr.

H. Kushida, Ikagaku-Kenkyujo, Kyoto. D - Mannurono - 3/- lactone was purchased from Sigma Chemical Company, St. Louis. Sodium L-guluronate was prepared as described previously (5). Chondroitin sulfate A was purchased from Seikagaku Kogyo Company, Tokyo. Heparin sodium salt from porcine intestine was heparin sample II, reported previously (4). Sodium hyaluronate and dermatan sulfate were prepared as described previously (4). The content of D-glucuronic acid in this whale dermatan sulfate was found to be 2.7% of total hexuronic acid by gas-chromatographic analysis. Sulfuric acid (98%, w/w) was purchased from Kokusan Chemical Company, Tokyo. Sodium tetraborate decahydrate and anhydrous sodium tetraborate were purchased from Koso Chemical Company, Tokyo, and Kanto Chemical Company, Tokyo, respectively. Other materials were commercial products. All reagents used were of analytical gra~le. Methods. The following procedures were used for quantitation of hexuronic acids: (i) the method of Dische (6), procedure a; (ii) the conventional procedure of Bitter and Muir using the reaction mixture consisting

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0003-2697/79/040295-04502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved.

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xlo' of 1 ml of a sample solution, 5.0 ml of 2O sulfuric acid, and 0.2 ml of 0.125% carbazole in absolute ethanol with 0.025 M NazB407" Man UA 10H~O (1), procedure b, or with 0.2 M 15 E Na~B4OT" 10H20, procedure c, or with 0.2 M E NazB4Oz.anhydrous, procedure d; (iii) the GulUA ~ 10 W small-scale procedure of Bitter and Muir fusing the reaction mixture consisting of 0.5 / ml of a sample solution, 3.0 ml of sulfuric acid, and 0.1 ml of 0.125% carbazole in 0 absolute ethanol with 0.025 M Na2B407. 0 0.05 0.10 0.15 0.20 10H20 (1), procedure e, or with 0.2 M MOLARITY OF NazB,,OT'IOH~O Na2B4OT" 10H20, procedure f, or with 0.2 M FIG. 2. Changes in extinction coefficients of the NazB4OT'anhydrous, procedure g. Unless color of D-mannuronolactone (ManUA) and Lotherwise stated, the small-scale procedure guluronate (GulUA) obtained by the small-scale B - M of Bitter and Muir (small-scale B - M methods in varying concentrations of borate (sodium method) with an indicated concentration of tetraborate decahydrate). borate was used. The reactions were carried out essentially as described by Bitter and Muir (1), except for slow addition of maximum intensity of color of D-glucuronosulfuric acid to a sample solution under lactone was obtained in the presence of 0.013 M borate. The color intensity decooling in an ice bath. creased very slightly with increasing concentration of borate. The color intensity of RESU LTS I=iduronolactone was minimum in the absence Changes in extinction coefficients of the of borate, but it increased rapidly up to a color of hexuronic acids obtained by the concentration of 0.05 M and reached a small-scale B - M methods in varying maximum at a concentration of 0.15 M. The concentrations of borate (sodium tetraborate difference in extinction coefficient of the decahydrate) are shown in Figs. 1 and 2. A color between D-glucuronolactone and Liduronolactone was smallest at a concentration of 0.2 M borate. The extinction coefX 10~ ficients of the color of o-mannuronolactone and L-guluronate, on the ordinate, plotted against concentration of borate, on the 15 ~ . ~ / IdUA abscissa (Fig. 2), were similar to the curve of L-iduronolactone. u~ The extinction coefficients of the color W ~o of t)-glucuronolactone obtained by procedures c, d, e, f, and g were compared to those obtained by procedure b (Table 1). It should be noted that the values obtained -d 005 "0i0 0.i5 o.io by these procedures were quite reproducible, MOLARITY OF Na,B,O¢IOH,O as far as the heating condition of the reacFIG. 1. Changes in extinction coefficients of the color tions in a boiling water bath maintained of D-glucuronolactone (GlcUA) and L-iduronolactone constant. Namely, changes in extinction (IdUA) obtained by the small-scale B - M methods in varying concentrations of borate (sodium tetraborate coefficients of the color of each authentic sample listed under Materials, obtained by decahydrate).

i/

297

QUANTITATION OF HEXURONIC ACIDS

several experiments in each procedure described above, were within + 1.5%. The extinction coefficient of the color obtained by procedure c was smaller by 12% as compared with that obtained by procedure b, and that obtained by procedure d was smaller by only 5%. As shown in Table 1, the ratio of the extinction coefficient of the color obtained by procedure b of Liduronolactone to that of D-glucuronolactone was 73%, whereas the ratios obtained by procedures c and d were as high as 97 and 96%, respectively. Similar results were obtained by procedures e, f, and g. The above observations indicated that procedure g is superior to procedures b and e thus far used for the quantitation of hexuronic acids, specifically for that of Liduronic acid. In procedure g, the extinction coefficients of the color given by a 1 M solution 1M (Ea~m,~z0nm) of D-glucuronolactone, Liduronolactone, D-mannuronolactone, and TABLE 1 EFFECT OF BORATE CONCENTRATION ON THE COLOR INTENSITY OF HEXURONIC ACIDS IN THE CARBAZOLE REACTION OF THE CONVENTIONAL (b, c, d) AND THE SMALL-SCALE (e, f, g) BITTER AND MUIR METHODS

Ratio o f E530 nm Procedure

GlcUA a

I d u A / G I c U A x 100b

b e

100 101

73 71

c f

88 91

97 95

d g

95 96

96 95

Ratios o f the extinction coefficients (Es~0 nrn) of the color of D-glucuronolactone (GlcUA) obtained by procedures c, d, e, f, and g to that obtained by procedure b. b Ratios of the extinction coefficients (E~30 nm) of the color of L-iduronolactone (IdUA) to that of D-glucuronolactone (GIcUA) u n d e r the above conditions.

TABLE 2 HEXURONIC ACID CONTENT OF GLYCOSAMINOGLYCANS DETERMINED BY THREE DIFFERENT PROCEDURES OF THE CARBAZOLE REACTION a

Procedure

Chondroitin sulfate A Hyaluronic acid Heparin D e r m a t a n sulfate

a

b

g

36.5 49.2 45.3 12.1

37.7 46.7 35.1 30.5

37,2 46.9 36.2 36.8

a Data are e x p r e s s e d as nag of D-glucuronic acid in 100 mg of the sample.

L-guluronate were 198 -- 2.1 x 102, 187 -- 1.8 x 102, 155-+ 1.8 x 102, and 118 x 1.2 x 102, respectively, in the final solution containing all the reagents. The extinction coefficient of the color of D-glucose obtained by procedure g was 12% of that of D-glucuronolactone. The hexuronic acid content of chondroitin sulfate A, hyaluronic acid, heparin, and dermatan sulfate was determined by procedures a (6), b (1), and g, respectively. As shown in Table 2, the latter two methods gave almost similar results with respect to hexuronic acid content in chondroitin sulfate A and hyaluronic acid. Of the three methods, however, procedure g gave the highest hexuronic acid content in dermatan sulfate, which was comparable to that in chondroitin sulfate A. Moreover, procedure g had almost the same advantages as procedure b, as far as the relationship between concentration and optical density, stability of color, reproducibility, and degree of interference from lmM NaIO4 or NaNO2 are concerned. DISCUSSION

The carbazole method of Bitter and Muir (1) has been used widely for quantitation of hexuronic acids. Blumenkrantz and Asboe-Hansen (7) recently developed an m-hydroxydiphenyl method. Their method

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exceeds the method of Bitter and Muir in sensitivity and specifity for uronic acid. However, the reagent is expensive. In this sense, the carbazole method still keeps its place. The color intensity of hexuronic acids or of glycosaminoglycans obtained by the original carbazole method (6) differs among one another (6,8). This experimental finding has been utilized for qualitative analysis of the components in a mixture of hexuronic acids (9-11). In the carbazole method of Bitter and Muir (1), the difference in color intensity of hexuronic acids of glycosaminoglycans among one another, except for heparin, is very small (1,2). However, this method is still unsatisfactory for the quantitation of hexuronic acids contained in dermatan sulfate (1,2) and in deamination products of heparin (3,4,12). In previous papers (3,4), we reported that the color intensity of L-iduronic acid and of the deamination products of heparin increased in the presence of 0.1 M sodium tetraborate decahydrate and also that the color intensity of D-glucuronic acid decreased (4). The rate of decrease in color intensity of Dglucuronic acid was very small when 0.2 M anhydrous sodium tetraborate was used as in procedure g. As indicated previously (13), the small amount of water introduced into the borate/sulfuric acid reagent from sodium tetraborate decahydrate is critical for the color reaction. When evaluated by procedure g, the hexuronic acid content of

dermatan sulfate was comparable to that of chondroitin sulfate A. The above observations indicated that procedure g is useful for quantitation of hexuronic acids in dermatan sulfate-type substances. ACKNOWLEDGMENTS We thank Dr. H. Kushida for supplying the material and Dr. M. Motomiya for his help in preparation of this paper. This work was supported by a grant from the Ministry of Education, Science and Culture of Japan.

REFERENCES l. Bitter, T., and Muir, H. M. (1962)Anal. Biochem. 4, 330-334. 2. Gregory, J. D. (1960)Arch. Biochem. Biophys. 89, 157-159. 3. Kosakai, M., and Yosizawa, Z. (1978)J. Biochem. 83, 1567-1575. 4. Kosakai, M., and Yosizawa, Z. (1978)J. Biochem. 84, 779-785. 5. Kosakai, M., and Yosizawa, Z. (1975) Anal. Biochem. 69, 415-419. 6. Dische, Z. (1947)J. Biol. Chem. 167, 189-198. 7. Blumenkrantz, N., and Asboe-Hansen, G. (1973) Anal. Biochem. 54, 484-489. 8. Dische, Z. (1955) Methods Biochem. Anal. 2, 342-358. 9. Hoffman, P., Linker, A., and Meyer, K. (1956) Science 124, 1552. 10. Hang, A., and Larsen, B. (1962) Acta Chem. Scand. 16, 1908-1918. 11. Kuntson, C. A., Jeanes, A. (1968)Anal. Biochem. 24, 482-490. 12. Lindahl, U., and Axelsson, O. (1971) J. Biol. Chem. 246, 74-82. 13. Kosakai, M., and Yosizawa, Z. (1978) Connective Tissue 9, 143-144.

A partial modification of the carbazole method of Bitter and Muir for quantitation of hexuronic acids.

ANALYTICAL BIOCHEMISTRY93, 295--298 (1979) A Partial Modification of the Carbazole Method of Bitter and Muir for Quantitation of Hexuronic Acids MASA...
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