ANALYTICAL BIOCHEMISTRY 32, 314-321 (1969)

A Modified Method for Chondrosulfatase Assay’

Y. KAWAI, N. SENO, AND K. ANN0

Department of Chemistry, Faculty of Science, Ochanomizu University, Bunkyo-ku, Tokyo, Japan

Received May 27, 1969

In assaying for sulfatases it is possible to use several methods which have been devised for the determination of inorganic sulfate. As a test for chondrosulfatase, the micro benzidine method developed by Dodgson and Spencer (1) is usually employed. This method appears to be the most suitable because of its good sensitivity and absence of substrate inter- ference, but its technique is complicated and demands a high degree of skill. On the other hand, the turbidimetric method of Dodgson and Price (2, 3), which has been used to determine inorganic sulfate liberated by acid hydrolysis, is simple and rapid, and has good reproducibility. How- ever, when this method is used for chondrosulfatase assay, unraded substrate chondroitin sulfates interfere by forming a cloudy complex with the gelatin reagent in the assay system.

In this study, the turbidimetric method was modified to permit deter- mination of inorganic sulfate in the presence of chondroitin sulfate, and used to assay the chondrosulfatase of squid liver.

MATERIALS AND METHODS

Barium chloride-gelatin reagent. Two kinds of reagent were prepared. Reagent A was the same as that described by Dodgson (2) : 0.5 gm of gelatin was dissolved in 100 ml hot water (6@-70°C) and, after standing at 4°C overnight, 0.5 gm barium chloride was added. The cloudy solution was allowed to stand for at least 2 hours at room temperature. Reagent B was prepared by increasing the quantity of gelatin to 1.0 gm in the same solution. These reagents could be used for one week if stored at 4°C.

Cetylpyridinium chloride (CPC) solution. Cetylpyridinium chloride (Tokyo Kasei Co. Ltd.) was dissolved in warm water (40°C) to the desired concentration. If a precipitate was formed, the solution was used after being warmed at 40°C.

Chondroitin sulfate A. Sodium chondroitin sulfate A was prepared from

IA preliminary report of this work was presented at the 39th General Meeting of the Japanese Biochemical Society, Seikagaku 38, 539 (1966).

314

CHONDROSULFATASE ASSAY 315

whale cartilage by the method of Meyer et al. (4), and converted to potassium salt according to the procedure of Dodgson et al. (5).

Spectroscopic measurements. These were made with a Hitachi spectro- photometer model 139 with glass cuvets and a 1 cm light path.

Preparation of squid liver enzyme. Acetone powder of the liver of Ommastrephes sloani pacificus was extracted with 0.2 M sodium acetate buffer, pH 7.0, and the extract was adjusted to pH 4.5 with 1.0 M acetic acid. After standing in a refrigerator overnight the precipitate was re- moved by centrifugation. The supernatant was adjusted to pH 5.0 with 1 M sodium acetate and used as the enzyme solution.

Determination of inorganic sulfate liberated by chondrosulfatase. The enzyme preparation was incubated with potassium chondroitin sulfate A at 37°C in 0.2 M sodium acetate buffer, pH 5.0, in a 10 ml centrifuge tube. At the end of incubation, cetylpyridinium chloride solution was added (3 mg CPC for each milligram of substrate) and the mixture was kept at 37°C for 15 minutes. The undigested chondroitin sulfate which pre- cipitated as a CP-complex was removed by centrifuging for 10 minutes at 2,000g. To the supernatant (0.2 ml, containing 10-200 pg SOL-), 3.8 ml 0.2 N hydrochloric acid and 1 ml barium chloride-gelatin Reagent B were added and the mixture was allowed to stand for 20 minutes at room temperature. The resultant turbidity was measured at 360 rnp or 500 rnp against a reagent blank prepared in t,he same way except that SO,- was omitted. A calibration curve was prepared with buffer solution of potas- sium sulfate.

Micro benzidine method. This was carried out according to Dodgson and Spencer (1).

RESULTS AND DISCUSSION

Removal of chondroitin sulfates. In order to apply the turbidimetric method to chondrosulfatase assay, the unraded chondroitin sulfate in the system should be removed, because the gelatin in the reagent forms a cloudy suspension with chondroitin sulfates. As a method to remove the chondroitin sulfates, ethanol precipitation was attempted. This method, however, did not succeed, since inorganic sulfate was also pre- cipitated with ethanol.

Cetylpyridinium chloride, however, could be used to precipitate the chondroitin sulfates as an insoluble complex. Cetylpyridinium chloride was added to the standard solution, and after centrifugation the sulfate content of the supernatant was estimated by the turbidimetric method. Although cetylpyridinium chloride appears to be advantageous as a rea- gent in that it removes only the chondroitin sulfates without precipitating inorganic sulfates, there remains the difficulty that cetylpyridinium chlo-

316 KAWAI, SENO, AND ANN0

ride forms precipitates with the trichloroacetic acid used as the reagent for sulfate assay. Therefore, the original turbidimetric method cannot be employed in this case.

Effects of acids. To modify the turbidimetric method so that it could be used in the presence of cetylpyridinium chloride, other acids with which trichloroacetic acid can be replaced were tested. The recoveries of added potassium sulfate from aqueous solution determined by the turbidimetric method using 3% acetic acid, 0.1, 0.2, and 0.5 N hydrochloric acid or no acid, in place of 3% trichloroacetic acid, are shown in Table 1. Good

TABLE 1 Effect of Acids on Recovery of Potassium Sulfate

Determined by the Turbidimetric Method

To a solution of KeS04 (0.2 ml) was added 3.8 ml of the indicated acids followed by 1 ml BaCl,-gelatin Reagent A. After 20 minutes the extinction at 500 mp was measured against the reagent blank.

SOa-- ion (fig)

Acid used Concn. Added Recovered

Control” Acetic acid HCl

No acid (water)

3% O.lN o.lN 0.2N 0.2N 0.5N

100 100 100 50

100 50

100 100

100 90 98 50 99 50

5 6

0 Determined by the original turbidimetric method (374 trichloroacetic acid).

recoveries of sulfate were obtained in both cases of 0.1 and 0.2 N hydro- chloric acid, whereas 3% acetic acid gave poor recovery. Little sulfate was recovered with 0.5 N hydrochloric acid or in the absence of acid.

The above results indicate that 0.1 and 0.2 N hydrochloric acid can be substituted for 3% trichloroacetic acid in determining sulfate by the turbidimetric method.

Effects of barium chloride-gelatin reagent and cetylpyridinium chloride. The effect of cetylpyridinium chloride on recovery of sulfate was tested by using two kinds of BaClz-gelatin reagent (Reagents A and B). When Reagent A was used in the system, as shown in Table 2, the recoveries of sulfate were reduced in all cases by addition of cetylpyridinium chloride. On the other hand, Reagent B gave good recoveries of sulfate with either 0.1 or 0.2 N hydrochloric acid, and there was little effect of cetylpyri- dinium chloride. Thus Reagent B and 0.2 N hydrochloric acid were chosen in this modification.

CHONDROSULFATASE ASSAY 317

TABLE 2 Effects of BaC$-Gelatin Reagents and Cetylpyridinium Chloride

on Recovery of Potassium Sulfate

To a solution of I&SO4 (0.2 ml) with or without CPC were added 3.8 ml of the indi- cated acid and 1 ml BaClzgelat,in Reagent. A or B. After 20 minutes t,he extinction at ,500 rnp was measured.

SOa-- ion (pg) BaClz-gelatin CPC

Acid used reagent” (mg/0.2 ml) Added Recovered

3% TCA A 0 100 100 B 0 100 101

O.lNHCl A 0 100 98 A 3 100 65 B 0 100 103 B 3 100 104 B 5 100 103 A 0 100 99 A 3 100 70 B 0 100 99 B 3 100 99

B 5 100 98

Q Preparation of the reagenk is described in the text,.

0.2,VHCI

Calibration curves of potassium sulfate in water and buffer solutions. Calibration curves of sulfate were prepared with the modified turbi- dimetric method described above in water and buffer solutions. Standard solutions of K&JO, in water and 0.2 M sodium acetate buffer, pH 5.0 (0.2 ml, containing 10-200 pg SO,- ions) were analyzed in the presence and absence of 5 mg cetylpyridinium chloride. As shown in Figure 1, greater sensitivity was achieved by measuring at 360 qp than at 500 mp, but good linearity was obtained at 500 m,~. No effect of cetylpyridinium chloride was observed in any case, but different curves were obtained for water and buffer solutions. This effect, an increase of the absorption coefficient in the sulfate estimation system, generally appeared in other buffer or various salt solutions of potassium sulfate, and was observed with the original turbidimetric method as well as the modified method.

Recovery of sulfate from enzyme-containing solutions. In order to test the recovery of sulfate from solutions containing squid liver enzyme, cetylpyridinium chloride, and chondroitin sulfate, the following procedure was used. The enzyme solution (0.5 ml) was incubated at 37°C with 0.2 ml of a solution of potassium sulfate in water or 0.2 N sodium acetate buffer, pH 5.0. After 1 hour, 0.5 ml 3% cetylpyridinium chloride solution and 0.5 ml 1% potassium chondroitin sulfate A were added and this mixture was kept at 37” for 15 minutes to precipitate completely the CP-

0.6

I

KAWAI, SENO, AND AN’N6

Fm. 1. Calibration curves for determination of inorganic sulfate by modified turbidimetric method. To 02 ml of K&O4 solution in water (left) and 0.2 M sodium acetate buffer, pH 5.0 (hht), containing 5 mg CPC (O), or not (a), were added 3.8 ml 0.2 N HCl followed by 1 ml Bach-gelatin Reagent B. After 20 minutes the extinctions at 360 and 500 rnp were measured against the reagent blank.

complex. After centrifugation, 0.2 ml supernatant was taken and the sulfate content was estimated by the modified turbidimetric method. Good recoveries were obtained in all cases (Table 3). The recovery of sulfate was not affected by presence of other polyanions, heparin, and ribonucleic acid.

TABLE 3 Recovery of Potassium Sulfate from Solutions Containing Squid Liver Enzyme,

Cetylpyridinium Chloride, and Chondroitin Sulfate

SO,-- ion (rg)

No. Incubation medium Added Recovered

Water 20 20 “ 50 49 “ 100 100 “ 200 197

0.2 M acetate buffer (pH 5.0) 20 21 ‘I 50 51 IL 100 100 ,‘ 200 200

CHONDROSULFATASE ASSAY 319

Assay of chondrosulfatase. The chondrosulfatase activity of squid liver enzyme was assayed by the modified turbidimetric method and the micro benzidine method. A tube containing 1.5 ml reaction mixture, including 5 mg potassium chondroitin sulfate A and 0.5 ml enzyme solution, was incubated at 37°C. At appropriate intervals, 0.1 ml reaction mixture from the tube was diluted to 2 ml with water, and 8 ml ethanol was added to it. After being kept in a refrigerator overnight, the mixture was centri- fuged and the inorganic sulfate of the supernatant was determined by t.he micro benzidine method. The remainder of the reaction mixture was tested by the modified turbidimetric method after removing undegraded substrate by adding 0.5 ml 3% cetylpyridinium chloride solution. Control determinations were run in which enzyme and substrate were separately incubated. As shown in Figure 2, chondrosulfatase activity was found in

250

6 12 1s 24

!CIME (hours)

7

FIG. 2. Chondrosulfatase activity of squid liver enzyme assayed by modified turbidimetric method (0) and the micro benzidine method (0). Incubation mixtures and assay methods are described in the text.

the squid liver enzyme by both methods. The fact that approximately the same values were obtained shows that the modified turbidimetric method can be used for chondrosulfatase assay. Although the sensitivity of this method is low as compared with that of the micro benzidine method, its simplicity, rapidity, and good reproducibility, as well as its applicability over a wide range of experimental conditions (2)) are highly advantageous for enzyme assay, particularly in the case of crude enzyme.

The occurrence of chondrosulfatase has been reported mainly in bac- teria (5-9) ; its presence in the liver of a marine mollusc, Charoniu Zam- pas, found in 1938 (10) was the first example of its occurrence in an

320 KAWAI, SENO, AND ANN0

animal and there was no report until the enzymes which liberate inor- ganic sulfate from chondroitin sulfates or sulfated oligosaccharides were recently obtained from bovine aorta (11)) viscera of Pate& vulgata (12), and rat liver (13). It has been reported that purified chondrosulfatase from Proteus vulgaris and Flavobacterium heparinum affect only specific sulfated disaccharide units and not polymer chondroitin sulfates (9, 14). If the squid liver chondrosulfatase has the same properties as bacterial enzymes, chondroitin sulfates may not be effective as substrates for the purified enzyme. The purification and further characterization of this enzyme is now proceeding.

The modified turbidimetric method can be also adapted to other cases, in which it is necessary to determine inorganic sulfate in the presence of acidic polymers. For example, in measuring the acid hydrolysis rate of ester sulfate of sulfated polysaccharides according to Rees (15), unde- graded polysaccharides will interfere if the turbidimetric method of Dodgson and Price is used, particularly at the initial stage of hydrolysis. This trouble may readily be avoided by using the modified method de- scribed in the present paper. This method has made it possible to deduce the sulfate positions of chondroitin sulfate E from squid cartilage (16,17).

SUMMARY

The turbidimetric method of Dodgson and Price has been modified for chondrosulfatase assay. The method involves removal of undegraded chondroitin sulfates with cetylpyridinium chloride, modification of the barium chloride-gelatin reagent, and substitution of 0.2 N hydrochloric acid for the 3% trichloroacetic acid of the original method.

By using this modified method, the presence of chondrosulfatase activ- ity was demonstrated in the liver of the squid, Ommastrephes sloani pacificus.

REFERENCES

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21, 506 (1956). 5. D~DGSON, K. S., LLOYD, A. G., AND SPENCER, B., Biochem. J. 65, 131 (1957). 6. NEUBERG, C., AND RUBIN, O., B&hem. 2.67, 82 (1914). 7. NEUBERQ, C., AND HOFFMAN, E., Biochem. 2. 234, 345 (1931). 8. PINWS, P., Nature 66, 187 (1950). 9. LINKER, A., HOFFMAN, P., MEYER, K., SAMPSON, P., AND KORN, E. D., J. Bid.

Chem. 235, 3061 (1960). 10. SODA, T., AND FAAMI, F., J. Chem. Sot. Japan 59, 1202 (1938). 11. HELD, E., AND BUDDECKE, E., 2. Physiol. C&m. 346, 1047 (1967).

CHONDROSULFATASE ASSAY 321

12. LLOYD, P. F., AND FIELDER. R. J., Biochem. .I. 109, 14p (1968). 13. TUDBALL, N., AND DAVIDSON, E. A., Biochim. Biophys. Acta 171, 113 (1969). 14. YAMAGATA, T., KAWAMURA, Y., AND SUZUKI, S., Biochim. Biophys. Acta 1

250 (1966). 15. REES, D. A., Biochem. J. 88, 343 (1963). 16. KAWAI, Y., SBNO, N., AND ANNO, K., J. B&hem. (Tokyo) 60, 317 (1966). 17. KAWAI, Y., Nat. Sci. Rept. Ochanomizu Univ. Tokyo 19, 23 (1968).

15,