/ . Biochem., 78, 1183-1190 (1975)

Amino Acid Composition and Physicochemical Characterization of Chondroitinase from Arlhrobacter aurescens Keiichiro HIYAMA and Shigetaka OKADA Department of Biochemistry, Osaka Municipal Technical Research Institute, Kita-ku, Osaka, Osaka 530 Received for publication, May 29, 1975

The amino acid and carbohydrate compositions of chondroitinase AC [EC 4.2.2.5] from Arthrobacter aurescens were determined, and its physicochemical properties were examined. 1. The enzyme has been shown to be a glycoprotein containing mannose, glucose, glucosamine, and glucuronic acid ( 3 : 5 : 4 : 2 ) . 2. Its molecular weight was estimated to be 76,000 by gel nitration on Sephadex G-200, 75,000-80,000 by SDS disc electrophoresis, and 75,800 by sedimentation velocity. No subunits were detected in the molecule. 3. The physicochemical properties determined include: sedimentation coefficient (s°20)W=5.14S), diffusion constant (£> 0 =6.09xl0- 7 cm2/sec), frictional ratio (f : fo=1.19) and apparent partial specific volume (y=0.73 ml/g). 4. The optical rotatory dispersion and circular dichroism of the enzyme were investigated. The contents of a-helix and ^-structure of the enzyme were estimated to be 16 and 25%, respectively.

The chondroitinase AC [chondroitin AC lyase, EC 4.2.2.5] of Arthrobacter aurescens has been crystallized and some properties of the enzyme were reported in a previous paper ( / ) . The enzyme is a lyase having a specificity similar to that of the chondroitinase of Flavobacterium heparinum, which was studied by Yamagata et al. (2). Studies on the amino acid composition and physicochemical properties of the two enzymes might provide an explanation for The abbreviations used in this report are SDS, sodium dodecyl sulfate; DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid); ORD, optical rotatory dispersion; CD, circular dichroism. Vol. 78, No. 6, 1975

the similarity of the specificity. Moreover, those results might provide data on the difference of specificity between the chondroitinases and hyaluronate lyases [EC 4.2.2.1]. However, there have been few reports on the amino acid composition and physicochemical properties of chondroitinase or hyaluronate lyase. Only Staphylococcus hyaluronate lyase has been studied as regards amino acid composition (3). Therefore, the amino acid composition of Arthrobacter chondroitinase was determined with an automatic amino acid analyzer, and some of the properties related to its size, shape, and molecular weight were examined. The optical rotatory power, which has been related

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K. HIYAMA and S. OKADA

to the a-helix content of soluble proteins, was also investigated. This paper presents the results of these studies. EXPERIMENTAL PROCEDURES Materials—The purified chondroitinase of Arthrobacter aurescens was prepared as described previously ( / ) . The standard proteins used for molecular weight estimation of the chondroitinase were: egg white lysozyme [EC 3.2.1.17] (14,200) from Eisai Co., ovalbumin (45,000) from Sigma Chem. Co., bovine serum albumin (68,000) from Sigma Chem. Co., Aspergillus niger glucose oxidase [EC 1.1.3.4] (152,000) from Kyowa Hakko Co. and catalase [EC 1.11.1.6] (250,000) from Kyowa Hakko Co. Gel Filtration—The method of Andrews ( 4 ) was followed for estimation of the molecular weight of the chondroitinase by gel filtration. A column of Sephadex G-200 (1.8x130 cm) elutedwith 0.05 M acetate buffer, pH 6.0, containing 0.1 M potassium chloride was used. Blue dextran was used to determine the void volume. SDS Polyacrylamide Gel Electrophoresis— The molecular weight of the chondroitinase was measured by SDS polyacrylamide gel electrophoresis according to the method of Weber and Osborn (5). Electrophoresis was performed in columns (0.48x8 cm) with 5.0% polyacrylamide gel at a constant current of 5 mA per gel for 2 hr. The markers used were egg white lysozyme, ovalbumin, bovine serum albumin, glucose oxidase, and catalase. Amino Add Analysis—The purified enzyme was lyophilized and dried to constant weight over P2O5 under reduced pressure. Accurately weighed portions (2.00 mg) of the enzyme were hydrolyzed with 2 ml of 6 M HC1 for 24, 48, and 72 hr at 110° in evacuated, sealed tubes. Amino acids were analyzed with a Hitachi KLA-3B automatic amino acid analyzer by the method of Spackman et al. (6). Tryptophan was determined by the method of Goodwin and Morton (7), and by the p-dimethylaminobenzaldehyde (p-DAB) method of Spies and Chambers (8). The amount of cysteine was estimated by sulfhydryl titration

according to the method of Ellman ( 9 ) . Carbohydrate Determination—The contents of neutral sugar and uronic acid in the enzyme were analyzed by the orcinol-sulfuric acid method of Winzler (10) and the carbazolesulfuric acid method of Bitter and Muir (11), respectively. For identification and quantitative analysis of each neutral sugar and uronic acid in the carbohydrate moiety, 10.0 mg of the enzyme was hydrolyzed with 2 ml of 1 M HC1 at 100° in an evacuated, sealed tube for 3 hr. After drying, the hydrolysate was dissolved in 1 ml of water and 0.5 ml of the solution was applied to a column (0.9x10 cm) of Dowex 50X-8, 200 to 400 mesh, H + form. The column was washed with water and 30 ml of the washings was evaporated to dryness on a rotary evaporator with a bath temperature of 35°. The dried sugars were dissolved in 1 ml of pyridine and reacted with N, O-bis(trimethylsilyl)trifluoroacetamide according to the method of Hoffman and Gooding (12). The reaction mixture was analyzed by gas-liquid chromatography. For the determination of amino sugars, the enzyme (10 mg) was hydrolyzed with 4 M HC1 in an evacuated, sealed tube at 100° for 4 hr. The hydrolysate was dried and dissolved in 1 ml of water, and then 0.5 ml of the solution was applied to a column of Dowex 50X-8. The amino sugars eluted with 2 M HC1 were subjected to trimethylsilylation and analyzed by gas-liquid chromatography. The remaining hydrolysates were used for a qualitative test by paper chromatography. Paper chromatography was carried out using Toyo No. 50 (40x40 cm) paper developed twice by the ascending technique in w-butanol : pyridine : water ( 6 : 4 : 3). The reducing sugars on the paper were detected by the silver nitrate method (13). They were identified by comparison with simultaneously run standards. Analytical Ultracentrifugation — Sedimentation velocity experiments were performed with a Hitachi Model UCA-1A ultracentrifuge (Hitachi Co., Ltd., Tokyo, Japan) at 20°, and followed by means of schlieren optics according to the procedure of Schachman (14), using a 12-mm double-sector cell at a rotor speed of 55,960 rpm. / . Biochem.

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ORD and CD Measurements—ORD and CD measurements were carried out at 20° with a Jasco ORD/UV-5 spectropolarimeter with a CD attachment. The dispersion constant (/fc) was calculated by means of the Drude equation (15). The Moffitt constants (a0 and bo) were calculated from the Moffitt-Yang equation (16). In calculations of reduced mean residue rotation ([m']) and molecular ellipticity ([0]), the mean residue weight was taken as 105, which was calculated from the amino acid composition. RESULTS Amino Acid and Carbohydrate Compositions—The amino acid composition of the purified chondroitinase is shown in Table I. Tryptophan content was calculated from the

absorbances of the enzyme at 290 and 295 nm, and from the absorbance at 600 nm by the pDAB method. Sulfhydryl titration with DTNB in the presence of 4.5 M guanidine hydrochloride showed that the enzyme has 16 SH groups per molecule. In the absence of the denaturing agent, about 2 SH groups per molecule reacted with DTNB at room temperature; the enzyme did not lose activity. The enzyme has high contents of alanine and glycine but a low content of methionine. The orcinol-sulfuric acid method indicated a neutral sugar content of 4.1% as glucose. Qualitative and quantitative studies by gasliquid chromatography showed that the carbohydrate moiety consists of mannose, glucose, glucosamine, and glucuronic acid in a ratio of 3 : 5 : 4 : 2 (Table II). These carbohydrate residues were also detected by paper chromatog-

TABLE I. Amino acid composition of chondroitinase from A. aurescens. See the text for details of the methods used. The data for 24, 48, and 72 hr are averages of two runs. Hydrolysis data rtcSlGUC

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ammonia Tryptophan Cysteine Total a

24 hr

48 hr

72 hr

24.4 14.1 42.1 77.2 73.9 57.8 56.5 29.7 108.6 120.4 70.2

mmole 25.7 15.4 43.5 82.2 73.1 56.3 59.3 29.9 109.1 123.8 71.8

30.3 15.8 41.8 80.8 70.6 53.3 58.0 27.9 102.6 120.4 73.8

9.2

6.7

3.2

29.2 79.0 23.0 26.4 44.3

29.7 78.4 20.1 26.0 49.0

30.3 76.6 14.2 24.9 63.3

48 hr or corrected value mmole 30.3" 15.8" 43.5 82.2 77.8=

60.3° 59.3 29.9 109.1 123.8 73.8" 10.5° 30.3" 79.01 23.7= 26.41 (44.3d) 22.4"

20.5'

Nfisrcst integer1

23 12 33 63 59 46 45 23 83 94 56 8 23 60 18 20

(34) 17 16 699

Calculated assuming that the methionine content is 8. " The values obtained after 72 hr hydrolysis were used. c Extrapolated to zero time, i The values obtained after 24 hr hydrolysis were used. e Determined photochemically (7) and by the p-DAB method (8). { Determined by the method of Ellman (9). Vol. 78, No. 6, 1975

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TABLE II. Carbohydrate composition of crystallized chondroitinase from A. aurescens. Carbohydrate moiety

Content (g/100g)

Molar ratio (moles/mole)

Mannose Glucose Glucosamine Glucuronic acid

0.64 1.05 0.81 0.46

3

1

ll

1

1

>

1

1

i

ill

i

i

Lysozyme

-

5 Ovalbumrn ^ L

4

2

raphy. No other amino sugars were detected, but glucuronic acid was found. Sedimentation and Diffusion Constants— The sedimentation velocity of the purified enzyme was measured at concentrations of 12, 8, and 4 mg of protein per ml, and gave values for 520,w of 4.96, 5.00, and 5.09xlO- 13 S, respectively. A iiner plot of these values, when extrapolated to zero concentration, gave •S20,w=5.14xl0-13 S. The material used for the sedimentation experiments was also used for diffusion studies. The diffusion constant, Do, was calculated by the height-area method (17), to be 6.09xl0~ 7 cm2 per second. Partial Specific Volume—The partial specific volume, zi0, was calculated, from the amino acid and carbohydrate compositions, to be 0.73 ml per g by the method of McMeekin et al. {18). Molecular Shape—The value s°=0.73 ml per g and S2 0jW =5.14xl0- 13 S were used to calculate a frictional ratio, f : f0 of 1.19. This low value indicates behavior approximating to that of an unsolvated sphere. Molecular Weight Estimation — The approximate molecular weight of the chondroitinase was estimated by the gel filtration method. A plot of elution volume per void volume against the logarithm of molecular weight is presented in Fig. 1. The chondroitinase was eluted between bovine serum albumin and glucose oxidase. From this result, an approximate molecular weight of 76,000±l,000 was obtained. SDS-polyacrylamide gel electrophoresis of the chondroitinase showed a single band. The approximate molecular weight of the enzyme was estimated from the mobility on SDS-poly-

Serum albumin

\ Chondroitinote —*•

2 -

-

Glucose oxidase

-

•C

•

Catalase

i

1

i

i

i

i

i

MOLECULAR

r

lit

5

10

i

20

i

30

WEIGHT ( X I O 4 )

Fig. 1. Molecular weight of chondroitinase by gel nitration on a calibrated Sephadex G-200 column. Chondroitinase and other standard proteins (egg white lysozyme, ovalbumin, bovine serum albumin, glucose oxidase, and catalase) were applied to a Sephadex G-200 column (1.8x130 cm) equilibrated and eluted with 0.01 M acetate buffer, pH 6.0, containing 0.1 M potassium chloride. Fractions of 2.0 ml were collected at a downward flow rate of 10 ml/hr. The open circle represents the elution point of chondroitinase activity.

acrylamide gel electrophoresis to be 75,000— 80,000 (Fig. 2). From these result, it was concluded that the chondroitinase has no subunits. Moreover, the molecular weight of the enzyme was calculated from the Svedberg equation, M=

RTS0 (l-6Vo)D o

where R, T, and po are the gas constant, temperature (Kelvin), and density of the solvent, respectively. This gave an estimate of 75,800 for the molecular weight, using the values of S2o,w (So), Do, and v° described above. ORD and CD—The ORD spectra of the enzyme in the absence and presence of 8 M urea are shown in Fig. 3. The ORD profile I. Biochem.

BACTERIAL CHONDROITINASE

1187

Catalase (native) ^Glucose onidase (native) .Catalase (dimer) Glucose oxidose (monomer) Serum albumin

talase (monomer)

Lysozyme

0 RELATIVE

0.5 MOBILITY

I

Fig. 2. Molecular weight of chondroitinase by electrophoresis in SDS-polyacrylamide gel. The logarithms of the molecular weights of the standard proteins were plotted against the relative mobility of bromophenol blue. The open circle shows the position of chondroitinase. Chondroitinase and marker proteins were incubated in 1% SDS-1% 2mercaptoethanol for 1 hr at 37°, respectively. The proteins were subjected to electrophoresis performed in a column (0.48x8 cm) with 5% polyacrylamide gel containing 0.1% SDS at a constant current of 5 mA per gel for 2 hr.

200

220

240

220 240 WAVELENGTH

Fig. 3. Ultraviolet ORD spectra of chondroitinase in the absence (solid line) and presence (broken line) of 8 M urea. The spectra were measured at pH 6.0, in a 1 mm quartz cell at protein cocentrations of 0.913 mg/ml (210-300 nm) and 0.457 mg/ml (195210 nm).

260

WAVELENGTH

{nm)

Fig. 4. Ultraviolet CD spectra of chondroitinase. The spectra were measured at pH 6.0, in 1 mm and 10 mm quartz cells at protein concentrations of 0.228 mg/ml (210-250 nm) or 0.114 mg/ml (190-210 nm), and 1.05 mg/ml (250-300 nm) for a and b, respectively. Closed circles are the calculated values for poly-L-lysine having 16% ar-helix, 25% ^-structure, and 59% random coil conformation. Vol. 78, No. 6, 1975

300

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K. HIYAMA and S. OKADA

tions—There have been few reports on the amino acid composition of chondroitinase, but some hyaluronidases have been studied. Staphylococcus hyaluronate lyase has a typical Solvent Ac an ba amino acid composition, with a large amount of basic amino acid residues (lysine; 48%, -211 Water 216 + 120 histidine; 34%) and cystine (or cysteine, 6%), -100 8 M urea 252 + 105 and a few hydrophobic amino acid residues (4.6%) (3). This is very different from the of the native enzyme in the 195—300 nm re- Arthrobacter chondroitinase (Table I), in spite gion exhibits a peak at 202 nm and a trough of the fact that they have the same hyaluronate at 232.5 nm. In the presence of 8 M urea, the lyase action. Table I shows that the residues ORD profile of the enzyme shows a trough at charged at pH 6 (aspartic acid, glutamic acid; 212 nm. To determine the dispersion constant lysine, arginine) amount to less than 23%, and from the Drude equation, [a] was plotted 40% can be classed as mainly hydrophobic in against X2[a\, and the value, Ac, was calculated nature (alanine, valine, leucine, isoleucine, from the slope of the line. The value /!c=216.4 methionine, phenylalanine, tyrosine). Therenm was obtained. In the calculation of the fore, this protein may offer an opportunity to Moffitt constants, Ao of the Moffitt-Yang equa- observe the effects of side chain interactions tion was set as 212 nm. From the intercept upon the ORD of native and denatured enzyme and slope of the plots ([m'](^ 2 /^ 0 2 -l) vs. proteins. l/(A2/A'02-D), a0 and b0 were calculated. These There have been many reports on enzymes data are summarized in Table III. to which a carbohydrate moiety is bound. Of The CD spectra of the enzyme are pre- various mucopolysaccharidases, a crystallized sented in Fig. 4. The value [0] was calculated testicular hyaluronidase [EC 3. 2.1.35] from a commercial preparation was reported to contain from the equation, N - acetylglucosamine (2.17%) and mannose [0]=3,300 4E L R (5.0%) (20), and Staphylococcus hyaluronate where 4SLR is the difference between the molar lyase was estimated to have 12—30% carboextinction coefficients for left- and right-circu- hydrate content (galactose : glucose : mannose = 1 : 3 : 6 , N - acetylglucosamine : N - acetyl larly polarized light. The CD profile of the enzyme in the 190—250 nm region exhibits a galactosamine=l : 1)(-?). However, the physpeak at 193 nm and troughs at 212 and 218 iological role of the carbohydrate moiety has not been elucidated in any instance. nm. The carbohydrate content in Arthrobacter The CD curve was analyzed according to the method of Greenfield and Fasman (19). chondroitinase is 4.1% as glucose, by the In Fig. 4, the closed circles show the calcu- orcinl-sulfuric acid method. Qualitative anallated values based on the 16% a-helix, 25% /3- yses of the carbohydrate moiety showed that structure, and 59% random coil conformation it consists of glucose, mannose, glucosamine of poly L-lysine. The observed CD curve fitted (possibly N-acetylglucosamine in the intact enthe calculated values well in the 215—240 nm zyme), and glucuronic acid. It is noteworthy that the carbohydrate composition is similar region. The CD spectrum in the 250-300 nm re- to that of the cell wall of Arthrobacter auresgion was very complicated, which is attributable cens (21). Because galactosamine was not deto the secondary structure and the environ- tected in the hydrolysate, it seems likely that the glucuronic acid in the hydrolysate does not ment of aromatic amino acid residues. arise from chondroitin sulfate. To confirm the existence of glucuronic acid in the enzyme DISCUSSION molecule, further investigations may be reAmino Acid and Carbohydrate Composi- quired, since few enzyme have been reported T A B L E III. Dispersion constants and Moffitt cons t a n t s of chondroitinase in the absence and presence of 8 M urea at pH 6.0.

/.

Biochem.

BACTERIAL CHONDROITINASE

to contain glucuronic acid. Molecular Weight—For globular proteins, the approximate molecular weight, M, is given by

The proportionality constant, k, was calculated using the published values (22) of s^o.w a n d M for hemoglobin, ovalbumin, and catalase. These proteins have frictional ratios close to that of chondroitinase, namely 1.14, 1.17, and 1.25, respectively. Values of k ranged from 2.806xlO"3 to 2.848 xlO' 3 (22), giving an average estimate of 77,500 for the molecular weight of the chondroitinase, using the 5.14S value for S2o,w This value agrees very closely with that of 76,000+1,000 estimated by gel filtration, SDS-polyacrylamide gel electrophoresis, and by calculation employing the Svedberg equation. Calculation of the k value using 76,000 as the molecular weight gave a value of 2.865 xlO" 3 . It is of interest that Arthrobacter chondroitinase has a molecular weight similar to the approximate value (70,000-80,000) for chondroitinase AC from F. heparinum (2), the.substrate specificity of which is the same as that of A. aurescens. Secondary Structure — There are various methods which can be used to examine the secondary structure of proteins. Riddiford (23) described methods to calculate the helical content of a protein using the optical rotatory values [m'] l99 =+70,200 and [m']Z33= -15,400 for native paramyosin, regarded as a 100% helical protein, and the value [m']233= — 2,090 ±20 for the random conformation of the protein. For native chondroitinase the values [m']i99= +8,100 and [m']233 = -2,830 were observed, and in 8 M urea the value [m']233 = —1,100 was obtained (Fig. 3). Calculating the a-helix content of the chondroitinase, values of 12% by the [m']im method and 13% by the [m']233 method were obtained. On the other hand, the helical content of the enzyme calculated by the b0 method was 17.7%, using the value bo=— 630 obtained for a perfect right-handed helix with poly(L-glutamic acid) (16) and the observed b0 values (Table III) for the enzyme. Vol. 78, No. 6. 1975

1189

Greenfield and Fasman proposed a method of examining the secondary structure of a protein in terms of the CD profile (19). In Fig. 4, the CD curve of chondroitinase in the 215—240 nm region fitted well with the calculated values based on the 16% a-helix, 25% ^-structure, and 59% random coil conformation of poly-L-lysine. The difference between the calculated and observed values below 215 nm may be caused by the effects of side-chains of amino acid residues or the carbohydrate moiety attached to the protein. The a-helix content estimated from the CD curve is in agreement with the averaged value based on the ORD data. According to Greenfield and Fasman (19), this method is superior to the methods utilizing ORD data. Therefore, it seems likely that chondroitinase has the 16% a-helix, 25% /3-structure, and 59% random coil conformation.

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1190 16. Moffitt, W. & Yang, J.T. (1956) Proc. Natl. Acad. Soc. U.S. 42, 596-603 17. Geddes, A.L. (1949) in Technique of Organic Chemistry, Physical Methods (Weissberger, A., ed.) Vol. 1, Part I, pp. 551, Interscience Publishers, Inc., New York 18. McMeekin, T.L., Groves, M.L., & Hipp, N.J. (1949) / . Am. Chem. Soc. 71, 3298-3300 19. Greenfield, N. & Fasman, G.D. (1969) Biochemistry 8, 4108-4116

K. HIYAMA and S. OKADA 20. Borders, C.L., Jr. & Raftely, M.A. (1968) / . Biol. Chem. 243, 3756-3762 21. Cummins, C.S. & Harris, H. (1959) Nature 184, 831-832 22. Tanford, C. (1967) in Physical Chemistry of Macromolecules pp.358, John Wiley and Sons, Inc., New York 23. Riddiford, L.M. (1966) /. Biol. Chem. 241, 27922802

/ . Biochem.