Purification and some properties of l -fucose dehydrogenase from Agrobacterium radiobacter and its application to the assay of bound-fucose in glycoconjugates

Biochimica et Biophysica Acta, l 117 (1992) 167-173 © 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4165/92/$05.00

167

BBAGEN 23709

Purification and some properties of L-fucose dehydrogenase from Agrobacterium radiobacter and its application to the assay of bound-fucose in glycoconjugates Y a s u n o b u Tsuji a, Ai Koike a, Kenji Y a m a m o t o b and Tatsurokuro Tochikura b " Research Laboratory of Higashimaru Shoyu Co., Ltd., Tominaga, Tatsuno, Tatsuno, Hyogo (Japan) and h Department of Food Science and Technology, Faculty of Agriculture. Kyoto Unicersity, Kitashirakawa, Sakyo-ku, Kyoto (Japan) (Received 24 February 1992)

Key words: L-Fucose dehydrogenase; a-L-Fucosidase; Bound-fucose assay; Agrobacterium radiobacter

L-Fucose dehydrogenase was found in the cell extract of Agrobacterium radiobacter and purified to homogeneity about 480-fold with 16% recovery. The molecular weight of the enzyme was approx. 64000. The enzyme was active in the neutral pH range, unlike other L-fucose or D-arabinose dehydrogenases which are active only in the alkaline pH range. Using this enzyme and a-L-fucosidase F-I of Bacillus circulans (Tsuji, Y., Yamamoto, K., Tochikura, T., Seno, T., Ohkubo, Y. and Yamaguchi, H. (1990) J. Biochem. 107, 324-330) simultaneously, we developed a new coupled enzymatic method in a single buffer system for determining bound-fucose in biological materials. The fucose released by a-L-fucosidase F-I was oxidized with L-fucose dehydrogenase in the presence of NAD +, and the NADH formed was measured by absorbance of ultraviolet or utilized to generate color in a reaction involving CuSO 4 and neocuproine. Using these methods, bound-fucose in various oligosaccharides and proteins such as lacto-N-fucopentaoses and porcine gastric mucin were quantitated within 15 rain.

Introduction L-Fucose is frequently located at the nonreducing termini of the carbohydrate moieties of several biologically active glycoconjugates, including serum glycoproteins, immunoglobulins, mucins, blood group substances, etc. [1]. Recently, accumulation of fucose-containing glycoconjugates has been reported in patients with cancer, cystic fibrosis and other diseases [2-5]. This fact indicates that the assay of bound-fucose may have an important diagnostic role. L-Fucose dehydrogenase (EC 1.1.1.122) catalyzes the oxidation of L-fucose to fucono-l,5-1actone using N A D + or N A D P + as a coenzyme. This enzyme has been purified from the livers of the rabbit [6], pig [7], and sheep [8], as well as cell extracts of Pseudomonas

Correspondence to: Y. Tsuji, Research Laboratory of Higashimaru Shoyo Co., Ltd., Tominaga, Tatsuno, Hyogo 679 41, Japan. Abbreviations: HPLC, high-performance liquid chromatography;Km, Michaelis constant; LNF, lacto-N-fucopentaose; PAGE, polyacrylamide gel electrophoresis; PGM, porcine gastric mucin; SDS, sodium dodecyl sulphate.

sp. [9] and Pullularia pullulans [10]. Some of them have been used for quantifying free L-fucose. However, limited supply of the enzyme has prevented it from being applied widely. Moreover, for the bound-fucose assay, these enzymatic procedures must be preceded by acid (or enzyme) treatment to hydrolyze the a-L-fucosidic bond to release free fucose [11,12]. We have previously reported that a-L-fucosidase F-I of Bacillus circulans isolated from soil had broad aglycon specificity and acted upon various intact macromolecules including porcine gastric mucin, various human erythrocytes, etc. We also showed that the enzyme was active in the p H range of 5.0 to 7.0. [13]. Recently, we found that Agrobacterium radiobacter produced large amounts of an L-fucose dehydrogenase which was active in the neutral p H range, unlike other enzymes which are active only at alkaline p H [6-10]. Using this L-fucose dehydrogenase and a-L-fucosidase F-I simultaneously, we developed a new coupled enzymatic method in a single buffer system for determining bound-fucose. This p a p e r describes the purification and some properties of L-fucose dehydrogenase from A. radiobacter, and a convenient and accurate method for

168 determining bound-fucose in various fucose-containing materials. Materials and Methods

Materials Lacto-N-fucopentaoses I, II, and III were purchased from BioCarb Chemicals, Lund, Sweden. NeocuproineHCI, porcine gastric mucin and hydroxylapatite were purchased from Nacalai Tesque, Kyoto, Japan. DEAE-Toyopearl 650C, Butyl-Toyopearl 650M, Toyopearl HW 55F, and TSK-Gel G3000SW were from Tosoh, Tokyo, Japan. 2'-Fucosyllactose, L-fucose, and L-fucose dehydrogenase (porcine liver) were obtained from Sigma, St. Louis, MO, USA. NAD + was purchased from Kohjin, Tokyo, Japan. All other chemicals used were of the highest grade available from commercial sources.

Enzyme assays L-Fucose dehydrogenase activity was assayed at 37°C by monitoring NAD + reduction at 340 nm. The reaction mixture, containing 625 /zl of 120 mM potassium phosphate buffer (pH 7.0), 50/zl of 15 mM NAD +, and 25 tzl of an appropriately diluted enzyme solution, was incubated at 37°C for 3 min. The reaction was started by adding 50 /~I of 150 mM L-fucose and the initial velocity was measured. One unit of enzyme activity was defined as the amount of enzyme which caused the reduction of 1 ~mol of NAD + per rain under the above assay conditions. a-L-Fucosidase activity was assayed using porcine gastric mucin as the substrate by the method described previously [13]. One unit of the enzyme activity was defined as the amount of the enzyme which released 1 /~mol of L-fucose per rain from the substrate.

Microorganism and cultit~ation Agrobacterium radiobacter IFO12607 used throughout this study was cultivated in medium containing 0.25% L-fucose, 0.5% peptone, 0.5% yeast extract and 0.5% NaC1 (pH 7.0). The cells were grown aerobically at 30°C for 20 h in a 10-1 jar fermentor (Mituwa Co.) containing 8 1 of the medium. The cells were harvested by centrifugation and stored frozen at -70°C.

for protein with Coomassie brilliant blue R. The positions of L-fucose, D-arabinose, and L-galactose dehydrogenase activities were located by staining the gels in a solution of 1 mM NAD +, 1 mM phenazine methosulfate, a trace of nitroblue tetrazolium, and 10 mM L-fucose, D-arabinose, and L-galactose, respectively [16]. Sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis was performed in 12.5% acrylamide and 0.1% SDS with a discontinuous Trisglycine buffer system by the method of Laemmli [17]. Phosphorylase b (mol wt 94 000), bovine serum albumin (mol wt 67000), ovalbumin (mol wt 43000), carbonic anhydrase (mol wt 30000), and trypsin inhibitor (tool wt 20 100) were used as molecular weight markers.

Molecular weight determination The molecular weight of the enzyme was determined using HPLC on a TSK-Gel G3000SW column (0.75 × 30 cm) equilibrated with 0.1 M potassium phosphate buffer (pH 7.0) containing 0.3 M NaCI. The column was calibrated with the following standard proteins (molecular weight in parentheses): bovine serum albumin (67000), ovalbumin (43000), carbonic anhydrase (29 000), and chymotrypsinogen A (25 000).

Purification of fucose dehydrogenase Unless otherwise indicated, purification of L-fucose dehydrogenase was performed at around 4°C and 10 mM potassium phosphate buffer (pH 7.0) was used. Centrifugation was carried out at 10000 × g for 20 to 30 min.

Step 1. Preparation of crude enzyme A. radiobacter cells (about 79 g, wet weight) were suspended in 395 ml of phosphate buffer and disrupted by passage through a French press (3 passages at 1500 kgf/cm2). The cell lysate was centrifuged (10000 × g, 10 min) and protamine sulfate was added to the supernatant (595 ml) to a final concentration of 0.16%. After standing for 30 min, the mixture was centrifuged (10000 × g, 20 min) and the precipitate was discarded. The supernatant obtained was used as a crude enzyme preparation.

Step 2. Ammonium sulfate fractionation

Protein was determined by the method of Lowry et al. [14], and from column chromatography fractions by the absorbance at 280 nm.

Solid ammonium sulfate was added to the crude enzyme preparation, and the 30 to 80% saturation precipitate was collected by centrifugation (10000 × g, 30 rain). The precipitate was dissolved in phosphate buffer and dialyzed overnight against the same buffer.

Gel electrophoresis

Step 3. DEAE-Toyopearl 650C column chromatography

Polyacrylamide disc gel electrophoresis was performed by the method of Davis [15] in 7.5% polyacrylamide with Tris-glycine buffer (pH 8.3), at a constant current of 2 mA per column at 4°C. Gels were stained

The dialyzed enzyme solution was applied to a DEAE-Toyopearl 650C column (3.5 × 37.5 cm) previously equilibrated with phosphate buffer. The column was thoroughly washed with the same buffer and the

Analyses

169 enzyme was eluted with a linear gradient of NaC1 in phosphate buffer (0 to 0.5 M). The active fractions were pooled and dialyzed overnight against phosphate buffer containing 15% (w/v) ammonium sulfate.

tions obtained from each application were pooled, concentrated by ultrafiltration, then dialyzed against phosphate buffer.

Preparation of a-L-fucosidase F-I Step 4. Butyl-Toyopearl 650M column chromatography The dialyzed enzyme solution was centrifuged and the supernatant was applied to a column (1.5 x 19 cm) of Butyl-Toyopearl 650M previously equilibrated with phosphate buffer containing 15% ammonium sulfate. The column was washed with the same buffer followed by elution with a reverse linear gradient of concentration of ammonium sulfate (15 to 0%). The active fractions were combined and concentrated by ultrafiltration through a membrane (PM-10; Amicon, Danvers, MA, USA).

Step 5. Toyopearl HW 55F gel filtration The concentrated enzyme solution was applied to a Toyopearl HW 55F column (1.5 X 120 cm, fractionation range (mol wt): 1000-700000) previously equilibrated with phosphate buffer and eluted with the same buffer. The active fractions were pooled.

Step 6. Hydroxylapatite column chromatography The enzyme solution was dialyzed against 1 mM phosphate buffer and eluted through a hydroxylapatite column (1.1 x 17 cm) previously equilibrated with the same phosphate buffer. The enzyme was eluted with a linear concentration gradient of phosphate buffer (1 to 250 mM). The active fractions were pooled, concentrated by ultrafiltration, and dialyzed against 0.1 M phosphate buffer containing 0.3 M NaCI.

a-L-Fucosidase F-I was prepared as described previously [13].

Determination of bound-fucose Unless otherwise indicated, the reaction mixture (0.75 ml) for ultraviolet detection contained the following: 100 mM potassium phosphate buffer (pH 7.0), 1 mM NAD +, 1.2 U of a-L-fucosidase F-I, 1.3 units of L-fucose dehydrogenase (0.01 ml), and the sample or L-fucose. Partially purified preparations of both enzymes are suitable for the assay. The blanks contained an identical mixture except that L-fucose dehydrogenase was replaced by 0.01 ml of water. The mixture was incubated at 37°C for 15 min and the formation of NADH was monitored from the increase in absorbance at 340 nm. Under these conditions, both reactions, including the hydrolysis of the sample and the oxidation of fucose proceeded to completion. For the colorimetric method, 0.75 ml of neocuproine-copper reagent comprising 1.48 mg of neocuproine-HCl and 0.57 mg of CuSO 4 in 0.2 M sodium acetate buffer (pH 4.7) was added to the above mixture after the 15 min incubation at 37°C. The resulting yellow color was measured at 450 nm by the method of Cohenford et al. [11]. Porcine gastric mucin was hydrolyzed with 2 M trifluoroacetic acid at 100°C for 3 h [18]. L-Fucose was determined using L-fucose dehydrogenase from porcine liver and the concentration was 0.17/xmol/mg.

Step 7. TSK-Gel G3OOOSWgel filtration on HPLC The dialyzed enzyme solution (1 ml) was repeatedly applied (0.1 ml volumes) to a gel filtration column (0.75 × 30 cm, fractionation range (mol wt): 5000500000) of TSK-Gel G3000SW (HPLC) previously equilibrated with 0.1 M phosphate buffer containing 0.3 M NaC1. The enzyme was eluted with the above buffer at a flow rate of 0.8 ml/min. The active frac-

Results

Purification of L-fucose dehydrogenase and purity of the enzyme Table I summarizes the results of the purification procedure. The overall recovery from the crude extract was about 16%, with about 480-fold purification.

TABLE I

Purification of L-fucose dehydrogenase Step

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Yield (%)

Fold purification (x)

Crude extract A m m o n i u m sulfate fractionation DEAE-Toyopearl 650C (CC * ) Butyl-Toyopearl 650M (CC * ) Toyopearl H W 55F (CC * ) Hydroxylapatite (CC * ) TSK-Gel 3000SW (HPLC)

9300 4000 390 38 9.2 3.9 3.0

1000 900 760 490 270 180 160

0.11 0.23 1.9 13.0 29.0 46.0 53.0

100 90 76 49 27 18 16

1 2.1 17.0 130 260 420 480

* Column chromatography.

170

~-100

galactose, o-mannose, D-fucose, etc.) had no effect on the oxidative reaction rate for L-fucose.

//>a

Substrate specificity

'~ 5(?, q, c~

; ' ;

i

I

10

pH

Fig. 1. Effect of pH on the enzyme activity. The enzyme activitywas assayed in 100 mM buffer under standard conditions. Buffer: ©, Mcllvaine; o, potassium phosphate; A Tris-HCl; A glycine-NaOH.

The purified enzyme migrated as a single protein band on native-polyacrylamide disc gel electrophoresis and its position coincided with that of L-fucose dehydrogenase activity. The D-arabinose and k-galactose dehydrogenase activity-stained bands also coincided with the protein band and the activity-stained band of L-fucose dehydrogenase.

L-Fucose, D-fucose, D-galactose, D-galactose, D-gluCOSe, D-mannose, L-arabinose, o-arabinose, L-xylose, D-xylose, and L-rhamnose were examined as possible substrates for L-fucose dehydrogenase under the standard assay conditions. The enzyme was active on Lfucose, D-arabinose, and L-galactose in the following order of velocity (relative activity in parentheses):Lfucose (100) > D-arabinose (71) > L-galactose (57). The other sugars were completely inactive. The apparent Michaelis constants (K m) for L-fucose and o-arabinose were calculated from Lineweaver-Burk plots to be 3.7 and 14.3 mM, respectively, at a fixed N A D + concentration of 5 mM. The enzyme was dependent on either NAD + or N A D P + as a coenzyme. The apparent K m values were 0.61 and 0.02 mM for N A D + and N A D P +, respectively, at a fixed t.-fucose concentration of 15 mM. The reaction velocity with N A D P + was, however, about 30% of that with N A D + for L-fucose dehydrogenation under the standard assay conditions.

Application of c-fucose dehydrogenase to the assay of Molecular weight

flgcose

The molecular weight of the enzyme was estimated to be about 64000 by gel filtration on TSK-Gel G3000SW (data not shown). SDS-PAGE of the purified enzyme gave two components with apparent tool wt of 37000 and 28000 indicating that the native enzyme contained two subunits.

The enzyme was used to assay free fucose. L-Fucose dehydrogenase (1.3 U) was allowed to react with known amounts of L-fucose under the standard assay conditions. The increase in absorbance at 340 nm for formed N A D H was proportional to the amount of L-fucose up to about 150 nmol. Similarly, a linear relationship between the absorbance at 450 nm with the neocuproine-copper reagent and the amount of fucose was observed. The standard curves yielded molecular absorption coefficients of 6.20 • 1 0 3 M i. c m - ~ for the ultraviolet method and 13.2. 1 0 3 M ~-cm -l for the colorimetric method.

Effects of pH and temperature on the enzyme activity and stability The enzyme was active in the pH range 7.0 to 9.0 (Fig. 1). The enzyme was stable in the pH range 7.5 to 9.5, whcn kept at 4°C for 69 h. The thermal stability of the enzyme was examined by heating it at various temperatures for 10 min. The enzyme was stable up to about 45°C. The enzyme could be stored frozen ( - 30°C) for over 6 months without any loss of activity, but repeated freezing and thawing lessened the activity.

Effects of various substances on the enzyme activity The effects of various sugars, metal ions, and SH-reagents on the enzyme activity were examined by preincubating the enzyme with the substances at 37°C for 15 min, followed by assaying the residual activity. Among the substances tested, Hg 2+ and Cu 2+ completely inhibited the enzyme activity at a concentration of 1 mM. p-Chloromercuric benzoate and N-ethylmaleimide (0.1 mM) strongly inhibited the enzyme activity. Other substances including various sugars (100 mM) tested (D-

Determination of bound-fucose in oligosaccharides The optimum pH of L-fucose dehydrogenase from

A. radiobacter was 7.5 to 8.0, whereas that of a-Lfucosidase F-I from B. circulans was 5.5 to 6.5. Since both enzymes had over 80% maximum activity at around pH 7.0, the coupled enzyme reaction in a single buffer system for determining bound-fucose was carried out at this pH. ee-t-fucosidase

F 1

Fucoconjugate ~ L-tucose+ afucoconjugate t -tucose

dehydrogcnase

L-Fucose+ NAD + 5 , L-fucono-1,_-lactone+ NADH+ H +

(1)

(2)

a-L-Fucosidase F-I (200 mU) and L-fucose dehydrogenase (250 mU) were incubated with 2'-fucosyllactose

171

/ 5O o

=#40

0 8 .k .

f

-

b

zlo

~

0.8

iv

/

g:20 ~L

Fig. 2. Fucose analysis of 2'-fucosyllactose by the proposed method with a-L-fucosidase F-I and L-fucose dehydrogenase. Reduction of N A D ÷ was measured by the ultraviolet method as described in the text. 2'-fucosyllactose (nmol): a, 6.25; b, 12.5; c, 25.0; d, 37.5; e, 50.0.

(6.25 to 50 nmol) under the standard assay conditions, then NAD+-reduction was monitored. As shown in Fig. 2, the increase in NAD+-reduction with each reaction mixture reached a plateau within 6 min and the level of NAD+-reduction was kept constant. The amounts of NADH formed coincided well with those of L-fucose included in the mixtures. Next, lacto-N-fucopentaoses I (a-(1 ~ 2)-L-fucosidic linkage), II (a-(1 ~ 4)-L-fucosidic linkage), and III (a(1 ~ 3)-L-fucosidic linkage) were used as samples under the above assay conditions and the results are summarized in Table II. Each reaction was completed within 15 min and the recovery of fucose was about 100%. The neocuproine-copper reagent was also applicable to this method.

T A B L E II

Determination of bound-fucose in lacto-N-fucopentaoses I (LNF-I), H (LNF-H) and III (LNF-III) Time required for completion

Total change of A340

Recovery (%)

(min)

Theoretical a

Observed

10.0 10.0 15.0

0.207 0.415 0.829

0.215 0.407 0.820

104 98 99

12.5 15.0 15.0

0.207 0.415 0.829

0.209 0.405 0.814

101 98 98

12.5 12.5 15.0

0.207 0.415 0.829

0.212 0.398 0.810

102 96 96

o6

. . . . . . . . iii

2

..... ~I

5 10 15 20 Reactiontime(rain)

+ ~'o 2 4 Reaction time (rain)

LNF-I 25 50 100 LNF-II 25 50 100 LNF-III 25 50 100

e

,'

•~ 30

Sample (nmol)

.

0.2

/

/

/

,/

oh ' o:3 ' o:~

PGM(mg)

Fig. 3. Fucose analysis of porcine gastric mucin by the proposed method. (a) (e . . . . . . e) Porcine gastric mucin (0.5 rag) was incubated with 1.3 U of a-L-fUcOsidase F-I. After incubation for the indicated time, the reaction was terminated by heating and the released fucose was assayed with 1.2 units of L-fucose dehydrogenase. O n the other hand, porcine gastric mucin (0.5 rag) was incubated with 1.3 units of a-L-fucosidase F-I and 0.15 (i), 0.75 (ii), 1.3 (iii) and 2.5 (iv) units of L-fucose dehydrogenase each under the standard assay conditions and then absorbance at 340 n m was monitored. (b) Various amounts of porcine gastric mucin (PGM) were used as samples under the standard assay conditions and the increase in absorbance at 340 nm was measured after a 15-rain incubation.

Determination of bound-fucose in porcine gastric mucin a-L-Fucosidase (1.2 U) was allowed to react with porcine gastric mucin (0.5 mg), a macromolecular glycoprotein, and the time course of fucose release from the glycoprotein was investigated (Fig. 3a). All L-fucose bound to porcine gastric mucin was completely liberated within 5 min and the amount coincided with that determined by acid hydrolysis. The effect of L-fucose dehydrogenase concentration on the rate of fucose oxidation was then investigated (Fig. 3a). The completion time for coupling reaction with 0.75, 1.3 and 2.5 U of L-fucose dehydrogenase was found to be about 15, 12.5 and 6 min, respectively. When porcine gastric mucin (0.1-0.5 mg) was incubated with 1.2 U of a-L-fucosidase and 1.3 U of L-fucose dehydrogenase, NAD+-reduction within 15 min proceeded linearly to the increase in the amount of porcin gastric mucin including L-fucose (Fig. 3b). Discussion

a The extinction coefficient of 6220 M - l . c m -1 was used for the calculation.

An NAD(P)+-dependent a-L-fucose dehydrogenase active in the neutral pH range has been isolated from A. radiobacter and characterized. The enzyme was active in the pH range 7.0 to 9.0. It was quite different from other L-fucose dehydrogenases which function optimally at pH 9.0 to 10.5 [6-10]. L-Fucose dehydrogenases [6-10] and D-arabinose dehydrogenases (EC

172 1.1.1.117) [19-21] oxidize L-fucose

O OH

71-t3 Ho/~H

H

D-arabinose

H H

O OH H

H H

a n d / o r L-galactose

H2OH O OH IHO ~ H

H

'

but the preferred substrate is L-fucose for the former enzymes and D-arabinose for the latter. The enzyme of A. radiobacter also utilized these sugars in the following order of velocity (relative activity in parentheses) : L-fucose (100) > o-arabinose (71) > L-galactose (57). The ratio of enzyme activities for these sugars during purification remained constant. Moreover, the final enzyme preparation migrated as a single P A G E protein band, which coincided with these three enzyme activities. Therefore, a single enzyme seemed to be possessed of three activities and the enzyme can be designated as L-fucose dehydrogenase. Three types of coenzyme-dependent L-fucose or Darabinose dehydrogenase are known. It was reported that D-arabinose dehydrogenases from Pseudomonas spp. [19,20] and pig liver [21] were active with both N A D + and N A D P +. e-Fucose dehydrogenase from Pseudomonas sp. was reported as having no activity with N A D + [9]. Mammalian L-fucose dehydrogenases [6-8] are active only with NAD+. The enzyme from A. radiobacter could utilize either N A D + or N A D P +, though the reaction velocity with N A D P + was slower than that with N A D +. To our knowledge, this is the first report of an L-fucose dehydrogenase active with both N A D + and NADP+. Glycoconjugates such as glycoproteins and glycolipids contain various sugars including L-fucose, but do not contain D-arabinose and L-galactose. Thus, the L-fucose dehydrogenase of A. radiobacter can be used to quantitate the fucose content of these complex substances. Even the crude preparation contained no activities of other sugar dehydrogenases except Darabinose and L-galactose dehydrogenases but only a

little activity of N A D + reductase. However, the reductase was not found after Butyl-Toyopearl 650M column chromatography (data not shown). Therefore, partially purified enzyme is also available for the assay. The first assay for determining bound-fucose described by Dische and Shettles [22] was insensitive and relatively cumbersome. Enzymatic methods using Lfucose dehydrogenase were then developed for the specific and reliable determination of free fucose. Cohenford et al. [11], Malar [12] and Grove et al. [23] have reported methods for quantitating bound-L-fucose in biological materials using L-fucose dehydrogenase. In their procedures, two reactions, hydrolysis of fucoconjugate to yield free fucose and fucose-oxidation with L-fucose dehydrogenase, were separately performed. They hydrolyzed fucoconjugate with 0.1 N HCI or 0.5 M H2SO 4 for 1-3 h at 100°C, or enzymatically with a-e-fucosidase for several hours at its optimum acid pH. After hydrolysis, the reaction mixture had to be neutralized for the subsequent action of L-fucose dehydrogenase. Therefore, these methods were very complex and time consuming. Furthermore, they could not be applied to native compounds without partially destroying them. The new method utilizes a-L-fucosidase F-I of B. circulans, which has broad aglycon specificity and Lfucose dehydrogenase of A. radiobacter simultaneously in a single buffer system at pH 7.0, at which both enzymes are active. It could be applied to the simple, rapid and effective quantitation of bound-L-fucose in various glycoconjugates. The coupling reactions were completed within 15 min. This method will contribute to the simplified diagnostic assay of- L-fucose, although some modifications may be necessary.

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173 14 Lowry, O.H., Rosebrough, N.J. Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 15 Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121,404-407. 16 Moore, R.O. and Virree, C.A. (1963) Science 142, 389-390. 17 Laemmli, U.K. (1970) Nature (London) 227, 680-685. 18 Lee, Y.C., Jhonson, G.S., White, B. and Scocca, J. (1971) Anal. Biochem. 43, 640-643.

19 Yamanaka, K. (1975) Agric. Biol. Chem. 39, 2227-2234. 20 Cline, A.L. and Hu, A.S.L. (1965) J. Biol. Chem. 240, 4488-4492. 21 Maijub, A.G.,. Pecht, M.A. and Carper, W.R. (1973) Biochim. Biophis. Acta, 37-42. 22 Dische, Z. and Sherries, L.B. (1948) J. Biol. Chem. 175, 595-603. 23 Grove, D.S. and Serif, G.S. (1981) Anal. Biochem. 111, 122-125.