3,4-Dinitrophenyl N-acetyl-β-d -glucosaminide, a synthetic substrate for direct spectrophotometric assay of N-acetyl-β-d -glucosaminidase or N-acetyl-β-d -hexosaminidase

ANALYTICALBIOCHEMISTRY

183,245-249

(1989)

3,4-Dinitrophenyl N-Acetyl-P-D-glucosaminide, a Synthetic Substrate for Direct Spectrophotometric Assay of N-Acetyl-P-o-glucosaminidase or N-Acetyl-P-D-hexosaminidase Tatsuhiko *Department and flatron

Received

May

Yagi,*

Ryuki

Hisada,*

and Hideto

Shibatat

of Chemistry and Chemical Education, Shizuoka University, Laboratories, Inc., Higashi-Kanda, Tokyo 101, Japan

836 @a, Shizuoka

422, Japan,

26,1939

3,4-Dinitrophenyl N-acetyl-B-D-glucosaminide (3,4dnpGlcNAc) was synthesized and proposed as an artificial substrate for N-acetyl-/3-D-glucosaminidase (EC 321.30) and IV - acetyl - @- D - hexosaminidase (EC 3.2.1.62) (collectively abbreviated as NAGase). 3,4dnpGlcNAc is water soluble and is fairly stable in aqueous medium. 3,4-Dinitrophenol released from 3,4-dnpGlcNAc by NAGase absorbs light of 400 nm at pH near 5, where the activity of urinary NAGase is assayed for diagnostic use. Direct and sensitive spectrophotometric assay of NAGase is, thus, possible using 3,4-dnpGlcNAc as an artificial substrate by monitoring the increase of Ah,,,, due to the release of 3,4-dinitrophenol in a thermostated spectrophotometer. 0 1889 Academic Press, Inc.

N-Acetyl-P-D-glucosaminidase (EC 3.2.1.30) and Nacetyl-/3-D-hexosaminidase (EC 3.2.1.52) (collectively abbreviated as NAGase)’ are present in mammalian organs such as kidney, liver mitochondria, spleen, and epidymis and catalyze hydrolytic release of an aglycon from N-acetyl$-D-glucosaminide (GlcNAc). The activity assay of the urinary NAGase is routinely carried out in the diagnosis of various kinds of renal disorders. One of the most prevailing assay methods consists of spectrophotometric determination of an aglycon rei Abbreviations used: NAGase, N-acetyl-fi-D-glucosaminidase (EC 3.2.1.30) and N-acetyl-@-D-hexosaminidase (EC 3.2.1.52); acetochloroglucosamine, Z-acetamido-2-deoxy-3,4,6-tri-o-acetyl-acopyranosyl chloride; np, nitrophenyl; GlcNAc, N-acetyl+D-glucosaminide; dnpGlcNAc, dinitrophenyl N-acetyl-P-D-glucosaminide; 4-npGlcNAc, 4-nitrophenyl N-acetyl-B-D-glucosaminide; A,,,,,, absorbance at 400 nm; EmMdw, millimolar absorption coefficient at 400 nm; U, pmol of aglycon released/min. 0003-2697/89$3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

leased from an artificial substrate such as 4-npGlcNAc (l-5). Since the optimum pH of the urinary NAGase is rather acidic and the released 4-nitrophenol develops color in an alkaline region, the enzymatic hydrolysis of the substrate is conducted at a pH near 5 for a definite period of time (e.g., 5 min), and then the reaction is terminated by the addition of alkali to measure the concentration of the 4-nitrophenolide ion spectrophotometritally. Direct spectrophotometric monitoring of the enzymatic release of aglycon from 4-npGlcNAc can be carried out in the ultraviolet region (6), but the millimolar absorption coefficient of the released aglycon is not sufficiently different from that of the substrate, and therefore the sensitivity of this assay method is not satisfactory. Several attempts have been made to synthesize new substrates of NAGase from which the release of aglycon is monitored directly and sensitively (7-10). These attempts, however, do not yet seemto be very successful. This paper reports advantageous characteristics of 3,4-dnpGlcNAc as an artificial substrate for a direct and sensitive spectrophotometric assay method of NAGase. MATERIALS

AND

METHODS

Purified NAGase from human placenta (6.1 mg protein/ml, 9.2 U/mg protein) was obtained from Sigma. The enzyme was diluted 600-fold in 1% bovine serum albumin containing 0.9% NaCl and stored in a cold room (4°C) as a stock solution. NMR spectra were recorded with a JEOL GSX-400 (400 MHz). The activity of NAGase was assayed by the standard method: 2.4 mM 4-npGlcNAc was used as an artificial substrate in 0.1 M citrate buffer, pH 4.5, at 37°C (5). 245

246

YAGI,

HISADA,

AND

SHIBATA

The activity was expressed in U (pm01 of aglycon released/min) . Hydrolytic release of 3,4-dinitrophenol from 3,4dnpGlcNAc by NAGase was monitored as follows: A reaction mixture containing 0.3 mmol citrate buffer, pH 5.0, and 3,4-dnpGlcNAc in 2.94 ml was placed in an optical cell (optical path, 10 mm) in a spectrophotometer thermostated at 37°C and the A400 was monitored. After 5 min of preincubation, 0.06 ml of the stock enzyme solution was added to the cell, the content was mixed immediately, and the monitoring of the AdO0 was continued to measure the rate of Ado,, increase per minute (dA/dt). From EmMJO,, of 3,4-dinitrophenol at the pH where the reaction was carried out (see below), the rate of hydrolysis of the substrate was calculated as follows, where the reaction rate is defined as micromoles of aglycon released per minute per milliliter of the reaction mixture:

6

J

260

reaction

rate = (dA/dt)/E,,

4oo.

SYNTHESIS

3,4-Dinitrophenol. It was synthesized by nitration of m-nitrophenol with 37% nitric acid followed by separation from dinitrophenol isomers and recrystallization from dry benzene (11): mp 133-135”C, pK, 5.40 (lit. mp 134”C, pK, 5.4). 3,4-Dinitrophenyl3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-P-D-glucopyranoside. An aqueous mixture of 3,4dinitrophenol (3.0 g, 16.3 mmol) and KOH (0.92 g) was thoroughly dried in vacua and refluxed with acetochloroglucosamine (Sigma, 7 g, 19 mmol) in 80 ml dry acetone for 3 h. The precipitated KC1 was removed and the acetone solution evaporated. The residue dissolved in chloroform was washed with 0.1 M NaOH and then with water several times. Upon evaporation of the chloroform solution, a white crystal mass of the condensation product was obtained. This was recrystallized from ethanol and dried in vacua: yield 5.9 g (11.4 mmol, 70%), mp 197201°C decomp. The proton NMR signals in CDC& with tetramethylsilane as an internal standard were assigned as follows (primed numbers for the aglycon): 6 = 1.96 (3H, S, N-acetyl), 2.08 (3H, S, 0-acetyl), 2.09 (3H, S, Oacetyl), 2.09 (3H, S, 0-acetyl), 3.99 (lH, ddd, J = 4.4,4.4, 9.5 Hz, H-5), 4.08 (lH, ddd, J = 8.1, 8.4, 10.3 Hz, H-2), 4.14-4.26 (2H, m, H-6), 5.11 (lH, t, J = 9.5 Hz, H-4), 5.49 (lH, dd J = 9.5, 10.3 Hz, H-3), 5.57 (lH, d, J = 8.1 Hz, H-l), 5.76 (lH, d, J = 8.4 Hz, NH), 7.26 (lH, dd, J = 2.6, 9.2 Hz, H-6’), 7.42 (lH, d, J = 2.6 Hz, H-2’), and 8.00 (lH, d, J = 9.2 Hz, H-5’). 3,4-Dinitrophenyl N-acetyl-/3-D-glucosaminide. The condensation product (3.76 g, 7.3 mmol) was dissolved in chloroform-dry methanol (40 ml each), O-deacetylated by dropwise addition of sodium methoxide (4 mmol/40 ml methanol) at O”C, and stirred for 60 min until turbid. The solution was concentrated in vacua to

340 WAVELENGTH,

400

460 NM

FIG. 1. Absorption spectra of 3,4-dnpGlcNAc (dotted line) and 3,4dinitrophenol (solid lines). The pH of the medium of 3,4-dinitrophenol is indicated in the figure.

half its volume, and the resulting white crystalline product was washed with chloroform and ethanol to remove the methoxide: yield 2.0 g (5.2 mmol, 70%), mp 144148°C decomp. The proton NMR signals of the O-deacetylated product in CD30D were slightly shifted from those of the condensation product in CDC4, except for the signals of H (amido) and H (0-acetyls), which disappeared. 2-Chloro-4-nitrophenyl N-ace@+D-glucosaminide. This glycoside was synthesized by condensing potassium 2-chloro-4-nitrophenolide and acetochloroglucosamine, followed by 0-deacetylation as described for 3,4-dnpGlcNAc. RESULTS

Solubility and spectral properties of 3,4-dnpGlcNAc. The solubility of 3,4dnpGlcNAc was determined to be 10.5 mM at 4°C whereas that of 2-chloro-4-npGlcNAc was only 2.0 mM. Figure 1 shows the spectra of 3,4-dnpGlcNAc and its aglycon, 3,4-dinitrophenol. They differ sufficiently from each other at the pH where the NAGase activity must be measured. EmM4ooof 3,4dinitrophenol at a given pH is expressed by the formula, 0.3 + 13.6/ (antilog(5.4-pH)+l), or specifically 0.3 at pH below 3, 4.2 at pH 5.0, 7.9 at pH 5.5, and 13.9 at pH over 8. The millimolar absorption coefficient of 3,4dnpGlcNAc is pH independent and is 5.9 at 280 nm, 6.2 at 290 nm, and 0.1 at 400 nm. Proportionality of the rate of hydrolysis of 3,4-dnpGlcNAc and the enzyme concentration. Figure 2 shows the time-course curves of the Ad00 change before and after

N-ACETYL-P-D-GLUCOSAMINIDASE

SPECTROPHOTOMETRIC

247

ASSAY

monitored at 400 nm was O.O14/min (i.e., O.O28/min when monitored in a cell of 10 mm optical path). The pH-activity curve using 3,4-dnpGlcNAc as a Figure 3 shows the relationship between the substrate. 0.8 pH and the rate of the enzymatic hydrolysis of 1.2 mM 3,4-dnpGlcNAc at 37°C. The pH optimum of NAGase was 4.5 (closed circles). The open circles in the figure 0.1 indicate the apparent enzymatic reaction rates expressed in dA/dt, i.e., there was no correction of the mea0.8 sured AdW for the increase in &M@& at higher pH (see Fig. 1). As can be seen in this figure the apparent enzymatic reaction rate was maximum at pH 5.4. Storage of 3,4-dnpGkNAc in aqueous media Solutions of 3,4dnpGlcNAc of different pHs were stored and the nonenzymatically released 3,4&nitrophenol was determined. The percentage of hydrolysis was 0.9% at 4°C during storage for 10 days and was pH independent between 3 and 10. An aqueous 3,4-dnpGlcNAc solution (1.2 mM in H20) stored for 5 days at 4°C (0.5% hydrolyzed) was nearly as effective as a freshly prepared 0.11 . ” ” ” . ‘. 0 2 4 8 solution as a substrate for the NAGase assay. However, t, min a 16-day-stored substrate solution (1.5% hydrolyzed) FIG. 2. Time-course curves of the A400 increase before and after the gave a reaction rate 90% of that given with the freshly addition of NAGase. The reaction mixture contained 0.1 M citrate prepared one. It is therefore recommended to use a subbuffer, pH 5.0, and 1.2 mM 3,4-dnpGlcNAc (final concentrations), and strate solution stored at 4°C within a week. Storage for the amount of NAGase added was (from the bottom) 0,2.13,4.25,6.38, 10 days at 10°C resulted in 2.3% hydrolysis of 3,4-dnp10.63, and 21.25 mu/ml reaction mixture, where mU is nmol aglycon released/min in the standard assay conditions with I-npGlcNAc at GlcNAc. pH 4.5 and at 37°C (5). The relation between dA/dt (the rate of the A400 Continuously monitored assay with 4-npGlcNAc as a increase/min) and the amount of NAGase contained in the reaction substrate. The apparent rate of the enzymatic hydrolymixture (mu/ml) is indicated in the insert. sis of 3,4-dnpGlcNAc catalyzed by 6.0 mu/ml NAGase A400

the addition

of different. amounts of NAGase to the reaccontaining 1.2 mM 3,4dnpGlcNAc in 0.1 M citrate buffer. The rate of the Adoo increase per minute (dA/dt) in the absence of NAGase at 37°C was 0.002 at pH 5. As shown in the insert of Fig. 2, the reaction rate is proportional to the amount of the enzyme added. K, of NAGase for 3,4-dnpGlcNAc. The rate of the enzymatic hydrolysis of 3,4-dnpGlcNAc (hereafter, enzymatic means that the rate of the hydrolysis was corrected for the nonenzymatic release of aglycon, see Fig. 2) was determined at various concentrations of the substrate at pH 5.0. The relationship between the reaction rate and the substrate concentration obeyed the Michaelis-Menten kinetics. The K, was determined to be 0.4 mM by the Lineweaver-Burk plot. Activity assay at the isosbestic wavelength of the released aglycon. The release of aglycon from 3,4-dnpGlcNAc was also monitored at 337 nm, where the millimolar absorption coefficient of the released aglycon is pH independent (Fig. 1). The procedure was identical to that mentioned above except that the reaction was carried out in a cell of 5 mm optical path. The apparent reaction rate (dA/dt) catalyzed by 5.6 U/ml of NAGase was 0.006 absorbance unit/min at 337 nm, whereas that tion

mixture

)rmol/min 0.005 dA /tit

w

2 0.004 a I

0.028 .0.024 8 -0.020 F

5; 0.003 . 2 : 0.002

.0.016

? 0

.

.0.008

I

70.004 JO.004 22

.0.012 a .

0.001

+

4

PH

G g

2 o

FIG. 3. The pH-activity curves of NAGase acting on 3,4-dnpGlcNAc. See the text for experimental details. The pH was measured at 18°C for the reaction mixture. Open circle: the apparent reaction rate (dA/dt) was measured by the AbW increase/min corrected for the control. Closed circle: the actual reaction rate (rmol aglycon released/ min/ml of the reaction mixture) was calculated from the apparent reaction rate divided by E,M 4W of 3,4-dinitrophenol at which the reaction was carried out. The amount of NAGase in the reaction mixture was 4.34 mu/ml, and the reaction temperature was 37-C.

248

YAGI, HISADA, AND SHIBATA

was 0.030 absorbance unit/min under the conditions described above. The release of aglycon catalyzed by 6.0 mu/ml NAGase was also monitored at 370 nm using 1.2 mM 4-npGlcNAc as a substrate (6). The rate of the AsTo increase was 0.005 absorbance unit/min. The apparent enzymatic reaction rate (dA/dt at 400 nm) with 3,4-dnpGlcNAc is, thus, six times as high as that (dA/dt at 370 nm) with 4-npGlcNAc as a substrate.

ciently distinguishable from that of the substrate at pH near 5. In the case of 4-nitrophenol (pK, = 7.21), a color develops upon ionization. An aglycon having enough color intensity at acidic pH must be a rather strong acid. Glycosides of GlcNAc with 2,4dinitrophenol (pK, = 4.0), 2-chloro-4-nitrophenol (pK,, = 5.45), and other halo-4-nitrophenols have been proposed as artificial substrates for the direct monitoring of NAGase (7-10). These substrates are, however, disadvantageous for routine uses. 2,4-dnpGlcNAc is synthesized with difficulty, DISCUSSION the overall yield from acetochloroglucosamine being There is an increasing demand for rapid and accurate only 5%) and is not stable in an aqueous medium during assay methods of diagnostic enzymes, and automated as- storage (7). 2-Chloro-4-npGlcNAc (8,9) can be synthesized in a better yield, but it is scarcely water soluble, the say systems are practiced in many biochemical laboratories in hospitals. In an ideal assay system, one has only solubility being only 2 mM. Some additives must, thereto add a reagent solution to a sample (blood, urine, or fore, be added to improve its solubility. 2-Fluoro-4other body fluids from a patient) in a thermostated spec- npGlcNAc is water soluble (lo), but its Km for NAGase is over 1 mM (calculated from the data presented in Ref. trophotometer to record the absorbance change automatically at a particular wavelength. The absorbance (lo)), and therefore a rather high substrate concentrachange to be determined is preferably an increase, for if tion is required to measure the activity accurately. 3,4-dnpGlcNAc described in this paper has the followthe absorbance decrease is to be measured, there will be a limit in the measurable substrate concentration. Typical ing advantageous properties for use in the direct monitoring of NAGase activity: (i) It can be synthesized in a examples are the activity assay systems for NAD-linked dehydrogenases where the rate of the Asa,, increase is re- reasonable yield (about 50%) from 3,4-dinitrophenol corded automatically upon the addition of the reagent and acetochloroglucosamine. The synthesis proceeds stoichiometrically, the loss being due to recrystallization (NAD+ and the substrate) to the sample (12). NAGase is an enzyme present in various animal tis- in small-scale experiments. (ii) It is water soluble. An sues. The urinary NAGase level increases in renal disor- aqueous solution of 10.5 mM 3,4-dnpGlcNAc can be preders, and a simple assay system for this enzyme is de- pared at 4°C. (iii) It is optically clear in a visible region. (iv) It is fairly stable in an aqueous medium. A substrate sired. 4-npGlcNAc is used as an artificial substrate for NAGase in current assay systems, in which the sub- solution stored for 5 days at 4°C which is 0.5% hydrostrate is incubated with the enzyme at an acidic pH for lyzed is as effective as a freshly prepared substrate solua definite period of time, the reaction is then stopped by tion for NAGase assay. (v) The rate of the nonenzymatic hydrolysis of 1.2 mM 3,4-dnpGlcNAc at 37°C is reproalkalinizing the incubation mixture, and 4nitrophenolide ion is measured spectrophotometrically at 400 nm ducible and is 0.002 absorbance unit/min at pH 5.0. Therefore only a few control runs are necessary in a (l-5). A fluorometric assay method using 4-methylumbelliferyl-GlcNAc as an alternate substrate (13,14) has series of routine assays. (vi) The reaction rate with 3,4an improved sensitivity, but the reaction must be dnpGlcNAc is 1.25 times that of 4-npGlcNAc at the optistopped to measure the fluorescence of the released mum pH, and the Km values of these substrates are similar, In other words, a more sensitive estimation of aglycon. A general assay method to directly monitor the release NAGase activity is possible using 3,4-dnpGlcNAc as a substrate. One might think of 3,4-dinitrophenyl N-aceof aglycon from 4-np-glycosides, catalyzed by any kind of glycosidases, has been proposed (6). This method, ap- tyl-/3-D-galactosaminide as another candidate for an artificial substrate, since galactosaminides are also known plied to the assay of NAGase, is based on the fact that EmMsdOof 4-nitrophenol is higher than Ern~ a40of 4- to be acted upon by NAGase. Since NAGase acts on ganpGlcNAc by 3.8. As the EmM 340of 4-npGlcNAc is 3.1 at lactosaminides more slowly with a lower pH optimum pH 5.0, the substrate concentration must be kept lower than on glucosaminides (2,3,5,7), use of the galactosamithan 0.6 mM. To measure the activity at a higher sub- nide does not seem advantageous. The NAGase reaction strate concentration, it is necessary to use a reaction cell can be monitored either at 337 nm, where the millimolar with a shorter optical path, or monitor the reaction at absorption coefficient of the released aglycon is pH inde370 nm, to lose the sensitivity of the assay in either case. pendent, or at 400 nm, where the assay is more sensitive. One possible disadvantage of 3,4dnpGlcNAc as a subIn order to measure the NAGase activity by direct and strate for NAGase would be the steep ErnMdWchange of continuous monitoring of the released aglycon, a subthe released aglycon near pH 5; i.e., a change of 0.1 pH strate must be optically transparent at the particular wavelength where the measurement is made, and the unit near pH 5 causes a 15% change of J&M400 of 3,4aglycon, when released, must have an absorbance suffi- dinitrophenol. This fact, however, need not be regarded

N-ACETYL-B-D-GLUCOSAMINIDASE

SPECTROPHOTOMETRIC

as a drawback. Figure 3 shows that the actual reaction rate of NAGase (closed circles) acting on 3,4-dnpGlcNAc is maximum at pH 4.5 as in the case of the standard procedure using 4-npGlcNAc (5) and is 50% active at pH 5.7. By increasing pH from 5 to 6 the activity decrease compensates with the absorbance increase, and therefore the apparent activity (open circles in Fig. 3) has a fairly flat plateau near pH 5.4. This means that strict control of pH is unnecessary when the pH of the reaction mixture is adjusted near 5.4. A possible assay procedure of NAGase is as follows: A mixture containing citrate buffer, pH near 5.4, and 3,4dnpGlcNAc, in a refrigerated reservoir, is automatically mixed with a urine sample and then transferred to an optical cell thermostated at 37°C and the rate of the AdO0increase (dA/dt) is recorded automatically. The concentrations of the citrate buffer and the substrate are adjusted so that their final concentrations after mixing are 0.1 M and 1.2 mM, respectively. The activity of NAGase expressed in U is calculated from dA/dt corrected for the nonenzymatic release of aglycon. We are indebted signal assignment. nical assistance.

to Dr. D. Uemura for NMR We thank Ms. Hiroko Sugiyama

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ACKNOWLEDGMENTS

249

ASSAY

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