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Rate assay of N-acetyl-β-d-hexosaminidase with 4-nitrophenyl N-acetyl-β-d-glucosaminide as an artificial substrate
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Clinica Chimica Acta 251 (1996) 53-64
Rate assay of N-acetyl-fl-D-hexosaminidase with ,4-nitrophenyl N-acetyl-fl-D-glucosaminide as an artificial substrate Hideto Shibata a, Tatsuhiko Yagi *b °latron Laboratory, Higashi-Kanda, Chiyoda-ku, Tokyo 101, Japan bDepartment of Chemistry, Shizuoka University, 836 Oya, Shizuoka 422, Japan Received 15 May 1995; revised 13 December 1995; accepted 15 December 1995
Abstract
A rapid and accurate rate assay method for N-acetyl-/~-D-hexosaminidase (EC 3.2.1.52, also known as N-acetyl-fl-D-giucosaminidase, or NAGase) using 4-nitrophenyl N-acetyl~-D-glucosaminide (NP-GlcNAc) as an artificial substrate was developed using diethylaminoethyl-~t-cyelodextrin (DEn-CD , where n is the number of diethylaminoethyl groups introduced to ct-cyclodextrin), as an additive to ionize 4-nitrophenol to yellowcolored 4-nitrophenoxide at pH near 5, where the enzyme acts optimally. A possible recipe for the rate assay of NAGase is as follows. Prepare a stock solution containing 4.8 mmol/l NP-GIcNAc and 1% DEn-CD (n is preferably near 17) in 0.1 mol/! glycolate buffer, pH 5.50. Introduce the stock solution and a properly diluted sample (urine or other body fluid) to a reaction cell placed in a spectrophotometer at a ratio of 1:1, and monitor the absorbance at 400 or 420 nm. The reaction rate (enzymatic activity) can be conveniently read directly from calibration plots prepared for a given lot of DE,-CD sample, or can be calculated from the rate of the absorbance increase, ionization degree of 4-nitrophenol at pH 5.50, and the millimolar absorbance coefficient of 4-nitrophenoxide in the presence of 0.5% DEn-CD.
Keywords: N-Acetyl-fl-D-hexosaminindase (EC 3.2.1.52); 4-Nitrophenyl N-acetyl-fl-Dglucosaminide; Rate assay; Diethylaminoethyl-~t-cyclodextrin
*Corresponding author. 0098-8981/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII $~009-8981(96)06292-4
54
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
1. Introduction N-acetyl-fl-D-hexosaminidase (EC 3.2.1.52, also known as N-acetyl-flD-glucosaminidase or NAGase) is present in mammalian organs such as kidney, liver, spleen, etc., and catalyzes hydrolytic release of an aglycon from N-acetyl-fl-D-glucosaminide (GlcNAc) or N-acetyl-fl-D-galactosaminide. The activity assay of the urinary NAGase is of diagnostic importance, because its activity rises at an early stage of renal disorder. Rapid and accurate assay of an enzyme can be achieved by the technique called rate assay, in which an enzyme solution to be assayed is simply mixed with a substrate solution in an optical cell, and the change in the absorbance at an appropriate wavelength is recorded by means of a recording spectrophotometer. 4-Nitrophenyl N-acetyl-fl-D-glucosaminide (NP-GlcNAc), routinely used as an artificial substrate for the end-point assay of NAGase, has been considered not to be usable for the rate assay, because the aglycon, 4-nitrophenol, released from the substrate is colorless and optically indistinguishable from the substrate at pH near 5, where the enzyme acts optimally. The standard assay method with this substrate involves the mixing of the enzyme with the substrate solution at pH near 5, the addition of alkali to stop the reaction, and at the same time to ionize the aglycon to 4-nitrophenoxide after a definite time interval (e.g. 5 min), and the measurement of the absorbance at 400 nm [-1-4-]. The strategy to design an artificial substrate for the rate assay of NAGase has been to synthesize an N-acetyl-fl-D-glucosaminide having an aglycon which is a rather strong acid and ionizes at pH low enough for the enzyme to be active, but such a glycoside may be unstable and difficult to synthesize (e.g. [5]). NP-GlcNAc could, however, be used as an artificial substrate if the released aglycon, 4-nitrophenol, is ionized at pH near 5 by means of an additive which promotes ionization of 4-nitrophenol. Diethylaminoethyle-cyclodextrin (DE-CD) is such an additive, which has been shown to promote ionization of 4-nitrophenol to 4-nitrophenoxide at pH near or below 5 ([-6], also shown in Fig. 1). This paper presents a new rate assay method of NAGase using NP-GIcNAc and DE-CD.
2. Materials and methods 2.1. Chemicals and urine samples Human placental NAGase (6.1 mg protein/ml, 9.2 units/ml) and NPGIcNAc were products of Sigma Chemical Co. The enzyme was diluted 600-fold in 1% bovine serum albumin containing 0.9% NaC1 and stored
14. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
55
0.5[ DE
(9 (3 r--
0.3
-2
0 JO
0.2
<:
2
4
6
8
10
pH Fig. 1. Effect of DE,-CD on the coloration due to ionization of 4-nitrophenol (0.02 mmol/l) in 0.05 mol/1 glycolate buffer. Curve N, coloration (A40o) of 4-nitrophenol in the absence of any additive; curve DE, coloration (A420) of 4-nitrophenol in the presence of 0.5% DE l ~-CD.
in a refrigerator as a stock solution. DE17-CD (DE,-CD of n = 17; n is the number of diethylaminoethyl groups introduced to ~CD) was prepared as described in [6]. Several urine samples from patients with various kinds of renal disorder (nephrosis, interstitial nephritis, diabetic nephrosis, etc.) were obtained from a hospital in Tokyo.
2.2. Standard assay method of NAGase The assay method in I-3] was used as the standard. The reaction mixture contained 2.4 mmol/l NP-GlcNAc in 0.1 mol/l citrate buffer, p H 4.5. The stock solution was preincubated at 37°C and mixed with the enzyme solution of appropriate dilution at time zero. The volume of the reaction mixture was 3.0 ml. After 5 rain of incubation, 1.0 ml of 1 mol/1 sodium carbonate was added to the reaction mixture to make the pH 10.2, and the concentration of the aglycon released was estimated by the absorbance at 400 n m (A4o0). The A4o o at time 0 (sodium carbonate was added simultaneously with the enzyme) was recorded, and subtracted from that after 5 min for every sample. The enzymatic activity was expressed in units (one unit of the enzyme releases 1 #mol of aglycon from the substrate per min).
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H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
2.3. Millimolar absorbance of 4-nitrophenoxide ion The millimolar absorbance of 4-nitrophenoxide is 18.6 at 400 nm in the absence of any cyclodextrin derivative. It changes little in the presence of DE,-CD of n smaller than 10. However, it changes significantly in the presence of DEIT-CD, and is 16.8 at 400 nm and 22.0 at 420 nm [6]. 3. Results
3.1. Choice of buffer solution for the rate assay In order to find a suitable buffer for the rate assay, the effect of the buffer solution to the enzymatic activity was estimated by the standard assay system, in which citrate (recommended in [3]) was replaced with acetate, glycolate, or succinate of the same pH. Table 1 illustrates that the enzyme acts poorly in acetate buffer, but acts well in citrate, succinate, or glycolate. Since the citrate and succinate are known to suppress the DEn-CDassisted ionization of 4-nitrophenol to 4-nitrophenoxide [6], they cannot be used for the rate assay with NP-GlcNAc as a substrate, in which the released 4-nitrophenol is quantitated by automatic spectrophotometry without interrupting the enzymatic reaction by the addition of alkali. The effect of glycolate on the DEn-CD-assisted ionization of 4-nitrophenol was, thus, tested by the method detailed in our previous paper [6], and the results are shown in Fig. 1. The pKa of 4-nitrophenol in 0.05 mol/1 glycolate buffer was estimated to be 5.40 in the presence of 0.5% DEIT-CD, whereas that in 0.1 mol/1 acetate buffer was 5.23 [6], i.e. DEI7-CD exerts nearly its full effect to lower pKa of 4-nitrophenol in 0.05 mol/l glycolate buffer. The pKa of 4-nitrophenol is 7.14 in the absence of any cyclodextrin derivatives (also shown in Fig. 1).
Table 1 Comparison of NAGase activity in various buffer solutions Buffer (pH 5.0)
Activity (nmol/min)
Relative activity (1.00 for citrate)
Acetate Glycolate Succinate Citrate
5.44 9.77 8.95 8.22
0.66 1.19 1.09 1.00
The enzymaticactivity was measured by the standard method except for the buffer used. See text for details.
1-1.Shibata, T. YagiI ClinicaChimicaActa 251 (1996) 53-64
57
3.2. Effect o f DEn-CD on the enzymatic activity As DE,-CD will be contained in the enzymatic reaction mixture to increase the ionization of 4-nitrophenol, the effect of some DE,-CDs on the enzymatic activity was estimated by the standard assay method. Table 2 indicates that DEn-CD of n smaller than 5 exerts no effect on the enzymatic activity, but DE17-CD was slightly inhibitory, and its effect must be calibrated in the rate assay. This table also gives the pKa values of 4-nitrophenol in 0.5% of various DE,-CDs. 3.3. ,Rate assay o f NAGase with NP-GIcNAc The reaction mixture containing 7.2 #mol NP-GIcNAc, 15 mg DE17CD, and 0.15 mmol glycolate buffer, pH 5.0, in 2.9 ml was placed in an optical cell (optical path; 10 mm) installed in a recording spectrophotometer, and preincubated for 5 min at 37°C. Then a 0.1-ml portion of a properly diluted NAGase solution was added to the mixture, and A ~ moniitored. The results are shown in Fig. 2. It is clearly shown that A ~ increases linearly with time, and the rate of A40o increase (AA40o/min) is proportional to the amount of NAGase added to the reaction mixture (the insert in the figure). It has been confirmed that A400 does not change at all under similar experimental conditions in the absence of any cyclodextrin derivatives (not shown).
3. 4. The apparent pH optimum and the real pH optimum The rate assay described in the preceding section was repeated except that the amount of NAGase was fixed, and the pH of the reaction mixture was changed. The results are shown in Fig. 3. Apparently the rate of A400 Table 2 Effect of DEn-CD on the activity of NAGase and on pKa of 4-nitrophenol Additive (0.5°/.)
Relative activity (1.00 for non-addition)
pK, of 4-nitrophenol
None DEo-CD DEs-CD DEIT-CD
1.00 1.00 1.02 0.70
7.14 6.32 5.78 5.40
The enzymatic activity was measured by the standard method, except that cyclodextrin derivative was contained in the reaction mixture as indicated, and the buffer(pH 5.0) used was 13.05mol/l glycolate.DEo-CD is non-substituted a-cyclodextrin.
58
H. Shibata, T. Yagi / Clinica Chimica Acta 251 (1996) 53-64
A 400 A:4OO.]min. × 100 0.14
0.12
0.10 |
-40
-20
!
0 Time
20
40
(s)
Fig. 2. Relationship between the rate of A4oo increase and the amount of NAGase contained in the reaction mixture. A mixture (2.9 ml) of 2.48 mmol/] NP-GlcNAc and 0.517% DE,v-CD in 0.0517 mol/l glycolate buffer, pH 5.0, was placed in an optical cell (optical path: l0 ram) in a recording spcctrophotometer at 37°C. An enzyme solution (0.1 ml) was added at the arrow, and the change in ,4400 was monitored. The enzyme added was (from the bottom of the figure) 0.00, 2.87, 5.74, 8.61, or 11.48 milliunits/ml of the reaction mixture. The inset indicates the relationship between AA4oo/min and the amount of the enzyme (milliunits/ml) contained in the reaction mixture.
increase is maximum at pH 5.7 (open circles). However, the actual reaction rates expressed in nmol/min (milliunits) must be calculated using the degrees of ionization of 4-nitrophenol at different pH values calculated from the pKa of 4-nitrophenol under the experimental conditions, as follows. degree of ionization = 1/{ 1 + exp[2.3026(pK,- pH)]} where pKa is 5.40 (see above). The pH-activity relationship thus calculated is shown in Fig. 3 (solid circles). The real pH optimum for NAGase is 4.8.
3.5. Kinetic parameters The rate assay of NAGase was repeated with the amount of NAGase
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
0 •
59
AA400/mill x 100 nmol/rain
// •
44
I
I
I
5
6
6.8
pH
Fig. 3. The pH- reaction rate relationship of NAGase. The reaction mixture contained 2.4 mmol/l NP-GIcNAc, 6.0 milliunits/ml of NAGase, and 0.5% DE17-CD in 0.05 mol/l glycolate buffer at pH between 4.58 and 6.70. Ordinate: the apparent reaction rate expressed in terms of AA~0o/min x 100 (O) and the actual rate of aglycon release expressed in terms of nmol/min per ml of the reaction mixture (.).
fixed and the pH of the reaction mixture at 5.50 (at which the ionization degree is 0.557), but the substrate concentration was varied. The results are shown in Fig. 4. From the Lineweaver-Burk plot (inset of the figure), the Km of NAGase for NP-GlcNAc was estimated to be 0.35 mmol/1, a value consistent with those reported for NAGases from other animal origins I-3,7,8]. This indicates that DE17-CD, although slightly inhibitory for the enzyme, does not have much interference for the binding of the enzyme to the substrate. 3.6. Comparison of the rate assay with the standard assay method The NAGase activities of urine samples from patients of various degrees of renal disorder were measured by the rate assay developed in this
60
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
V-I
.
.~
.
.
.
2
4
I-
1 2 substrate concentration
'S ol
s (raM)
Fig. 4. The relationship between the reaction rate (v, in nmol/min per ml of the reaction mixture) and the concentration of NP-GIcNAc (s, in retool/l). The reaction mixture contained 4.7 milliunits/ml of NAGase, 0.5% D E I 7 - C D , and varying concentrations of NP-GlcNAc in 0.05 mol/l glycolate buffer, pH 5.50, at which the degree of ionization of 4-nitrophenol is 0.557. The inset is the Lineweaver-Burkplot.
research and by the standard assay m e t h o d [3] at the same time, and the activities of both methods are compared as shown in Fig. 5. The correlation coefficient r between the data from these two methods was 0.991.
3. 7. Reliability and reproducibility o f the rate assay method Each of two urine samples, one from a nephritic patient and the other from a healthy individual, was divided into 5 portions and stored frozen at - 8 0 ° C . At intervals, the frozen samples were taken out, and the NAGase activities were determined by the rate assay method. The results are given in Table 3.
4. Discussion For the routine assay of enzymatic activities of biological fluids, the advantage of the rate assay over the end-point assay based on either spectrophotometry or spectrofluorometry is obvious. In the end-point assay, the operator has to measure the absorbance or fluorescence at least twice, at time zero and after a definite time interval for every sample,
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
61
0.06
0.04 .c_
o
o
'~
o
0.02
°o° o
o
o
0.00
,
i 2
,
, 4
,
, 6
, 8
n m o l / m i n p e r m l of t h e r e a c t i o n m i x t u r e
Fig. 5. Relationship between the NAGase activities measured by the rate assay method and those assayed by the standard method. The rate assay was conducted with 2.4 mmol/I NP-GIcNAc and 0.5% DE17-CD in 0.05 mol/l glycolate buffer, pH 5.5, at 37°C, and the observed rate of A42o increase (AA42o/min) was plotted (ordinate) against the enzymatic activity (nmol/min per mi of the reaction mixture) measured by the standard assay (abscissa). The number of samples was 18:15 from patients of renal disorder, 1 from a healthy individual, 1 (e located upper right) from human placenta (product of Sigma Chemical Co.), and 1 (e near the origin) for the control without enzyme. The correlation coefficiient r was 0.991.
because biological fluids such as urines or sera may be colored differently, and may contain fluorescent or fluorescence-quenching substances. A simple and reliable rate assay method is desired to meet an increasing demand for the activity assay of enzymes of diagnostic importance. Urinary NAGase is one of the diagnostic enzymes that increases at an early stage of various kinds of renal disorder. Several artificial substrates are proposed for the assay of this enzyme. NP-GIcNAc is one of the most prevalent substrates for the end-point assay of this enzyme: this substrate is easy to synthesize and commercially available, soluble in water, and its aqueous solution can be stored without decomposition. It is, however, difficult to conduct a rate assay with this substrate, because the aglycon 4-nitrophenol released from this substrate is rather a weak acid, and does not ionize to be spectrally distinguishable from the substrate at low pH, where: the enzyme acts optimally. Restricting our discussion to the artificial substrates for NAGase to be of N-acetyl-fl-D-glucosaminides of substituted phenols, rate assay may be
62
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
Table 3 Reproducibility of the rate assay method applied to urine samples Storage after sampling (days)
2 6 9 13 16
No. of measurements n
NAGase activity (AA420/min) Urine from a nephritic patient
Urine from a healthy individual
Activity
a
Activity
a
10 10 10 10 10
0.04778 0.04845 0.04828 0.04834 0.04798
0.00065 0.00093 0.00091 0.00106 0.00095
0.00735 0.00736 0.00714 0.00714 0.00752
0.00033 0.00032 0.00026 0.00011 0.00028
50
0.04817
0.00095
0.00730
0.00031
The rate assay was conducted with 2.4 mmol/1 NP-GIcNAc and 0.5% DE17-CD in 0.05 mol/l glycolate buffer, pH 5.5, at 37°C, and the rate of A42o increase (AA42o/min) was measured in an optical cell (optical path: 10 mm). Each reaction mixture contained 0.1 volume of the urine sample. Using Fig. 5, the observed rate of A,2 o increase (AA,2o/ min --- 0.04817) with the urine sample from the nephritic patient was converted to the reaction rate of 6.67 nmol/min per ml of the reaction mixture, i.e. 66.7 nmol/min per ml of the urine sample. Similarly, the NAGase activity of the urine sample from the healthy individual was estimated to be 7.9 nmol/min per ml of the urine sample.
possible if the aglycon of the substrate is rather a strong acid and ionizes upon enzymatic release at pH near 5. Such artificial substrates proposed for practical uses include 2-chloro-4-nitrophenyl fl-GlcNAc (I) [9-1, 3,4dinitrophenyl fl-GlcNAc (II) [10], 2-fluoro-4-nitrophenyl ~-GlcNAc (III) [11], etc. The pKa of the aglycons from substrates I and II are 5.45 and 5.38, respectively. A disadvantage of I is its low solubility: the reaction mixture containing this substrate must be supplemented with complicated additives to improve its solubility for use in the rate assay [9]. II is much more soluble, but slowly decomposes in solution upon storage at 4°C. It is recommended to use its stock solution within a week [10]. A disadvantage of Ill is its rather high Km. The rate assay method presented in this paper solves these problems. NP-GlcNAc is commercially available, sufficiently soluble, and its solution can be stored in a refrigerator for a year without appreciable decomposition. The aglycon released from NP-GlcNAc is, by itself, a weak acid (hence its glycoside is chemically stable), but behaves like a stronger acid to develop a yellow color at lower pH in the presence of DEI7-CD, whose
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
63
effect on NAGase is marginal. Synthesis of D E , - C D of n near 17 may be laborious [6], but does not require any special technique of organic chemistry. It is advisable to synthesize DE,-CD of n near 17 in bulk to evaluate experimentally its ability of lowering pKa of 4-nitrophenol and its effect on the activity of NAGase under the assay conditions before its use in the rate assay of NAGase. The new rate assay method proposed in this research was shown to be reliable and reproducible (Table 3), and gave comparable results to those obtained by the standard assay method as shown in Fig. 5. This research was conducted with DE17-CD which had been prepared in bulk, but DEn-CD of n < 17 may be similarly employed. As every DE,-CD preparation with different n may promote ionization of 4-nitrophenol differently I-6], its effect on lowering pKa of 4-nitrophenol in the reaction mixture must be estimated for every lot of preparation. For example, DEs-CD will be used as an additive for the rate assay, but the sensitivity with this additive may be a little lower, because the pK a of 4-nitrophenol with this additive is 5.78 (Table 2), and hence the degree of ionization is calculated to be 0.345 at pH 5.5. A possible recipe for the rate assay of NAGase is as follows. Prepare a stock solution containing 4.8 mmol/l NP-GlcNAc and 1% DE17-CD in 0.1 mol/1 glycolate buffer, pH 5.50, a non-inhibitory aseptic being added if neces,;ary. Introduce the stock solution and a properly diluted sample (urine or other body fluid) to a reaction cell placed in a spectrophotometer at a ratio of 1:1, and monitor A400 or A420. The ratio of the stock solution and tile urine sample may be variable as far as the composition of the final mixture is kept unchanged. The reaction rate (enzymatic activity) can be conveniently read directly from calibration plots as shown in Fig. 5 (such a figure must be prepared for a given lot of DE.-CD sample), or can be calculated from the rate of the absorbance increase (AA400/min or AA420/ min, ionization degree of 4-nitrophenol at pH 5.50, and the millimolar absorbance coefficient of 4-nitrophenoxide in the presence of 0.5% DE17CD (16.8 at 400 nm or 22.0 at 420 nm).
References
[1] Li YT, Li SC. ~t-Mannosidase,fl-N-acetyl-hexosaminidase,and fl-galactosidasefrom jack bean meal. Methods Enzymol 1972;28:702-713. [2] AgrawalKM L, Bahl OP. a-Galactosidase,fl-galactosidase,fl-glucosidase,fl-N-acetyigilucosaminidase, and ~t-mannosidase from pinto beans. Methods Enzymol 1972;28:720-728. [31 Tarentino AL, Maley F. fl-N-Acetylglucosaminidasefrom hen oviduct. Methods Enzymoi 1972;28:772-776.
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H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
[4] McGuire EJ, Chipowsky S, Roseman S. fl-N-Acetylglucosaminidase, ~t-N-acetylgalactosaminidase, and fl- galactosidase from Clostridium perfringens. Methods Enzymol 1972;28:755-763. I'5] Jones CS, Kosman DJ. Purification, properties, kinetics, and mechanism of E-Nacetylglucosaminidase from Aspergillus niger. J Biol Chem 1980;255:11861-11 869. 1'6] Yagi T, Aoshima R, Kuwahara M, Shibata H. Preferential inclusion of nitrophenoxide ion over its conjugate acid in polycationic cyclodextrin: pK, decrease of nitrophenols by host-guest interaction with diethylaminoethyl-cyclodextrins. J Inclusion Phenom 1993;16:231-243. [7] Frohwein YZ, Gatt S. Isolation of fl-N-acetylhexosaminidase, fl-N-acetylglucosaminidase, and fl-N-acetylgalactosaminidase from calf brain. Biochemistry 1967; 6:2775-2782. 1-8] Geiger B, Calef E, Arnon R. Biochemical and immunochemical characterization of hexosaminidase P. Biochemistry 1978;17:1713-1717. 1-9] Makise J, Ichikawa K, Yoshida K, Watanabe S. New derivatives of glucosaminide and the assay reagents using these derivatives as substrates for N-acetyl-fl-oglucosaminidase. Patent 1742610, Japan, 1993. [I0-1 Yagi T, Hisada R, Shibata H. 3,4-Dinitrophenyl N-acetyl-fl-o-glucosaminide, a synthetic substrate for direct spectrophotometric assay of N-acetyl-fl-o-glucosaminidase or N-acetyl-fl-D-hexosamirndase. Anal Biochem 1989;183:245-249. [111 Kurihara T, Kamimura M, Hayashi Y, Teshima S. Derivatives of N-acetyl-fl-oglucosaminide and the assay reagents using these derivatives as substrates for Nacetyl-fl-D-hexosaminidase. Patent 1847729, Japan, 1994.
Clinica Chimica Acta 251 (1996) 53-64
Rate assay of N-acetyl-fl-D-hexosaminidase with ,4-nitrophenyl N-acetyl-fl-D-glucosaminide as an artificial substrate Hideto Shibata a, Tatsuhiko Yagi *b °latron Laboratory, Higashi-Kanda, Chiyoda-ku, Tokyo 101, Japan bDepartment of Chemistry, Shizuoka University, 836 Oya, Shizuoka 422, Japan Received 15 May 1995; revised 13 December 1995; accepted 15 December 1995
Abstract
A rapid and accurate rate assay method for N-acetyl-/~-D-hexosaminidase (EC 3.2.1.52, also known as N-acetyl-fl-D-giucosaminidase, or NAGase) using 4-nitrophenyl N-acetyl~-D-glucosaminide (NP-GlcNAc) as an artificial substrate was developed using diethylaminoethyl-~t-cyelodextrin (DEn-CD , where n is the number of diethylaminoethyl groups introduced to ct-cyclodextrin), as an additive to ionize 4-nitrophenol to yellowcolored 4-nitrophenoxide at pH near 5, where the enzyme acts optimally. A possible recipe for the rate assay of NAGase is as follows. Prepare a stock solution containing 4.8 mmol/l NP-GIcNAc and 1% DEn-CD (n is preferably near 17) in 0.1 mol/! glycolate buffer, pH 5.50. Introduce the stock solution and a properly diluted sample (urine or other body fluid) to a reaction cell placed in a spectrophotometer at a ratio of 1:1, and monitor the absorbance at 400 or 420 nm. The reaction rate (enzymatic activity) can be conveniently read directly from calibration plots prepared for a given lot of DE,-CD sample, or can be calculated from the rate of the absorbance increase, ionization degree of 4-nitrophenol at pH 5.50, and the millimolar absorbance coefficient of 4-nitrophenoxide in the presence of 0.5% DEn-CD.
Keywords: N-Acetyl-fl-D-hexosaminindase (EC 3.2.1.52); 4-Nitrophenyl N-acetyl-fl-Dglucosaminide; Rate assay; Diethylaminoethyl-~t-cyclodextrin
*Corresponding author. 0098-8981/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII $~009-8981(96)06292-4
54
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
1. Introduction N-acetyl-fl-D-hexosaminidase (EC 3.2.1.52, also known as N-acetyl-flD-glucosaminidase or NAGase) is present in mammalian organs such as kidney, liver, spleen, etc., and catalyzes hydrolytic release of an aglycon from N-acetyl-fl-D-glucosaminide (GlcNAc) or N-acetyl-fl-D-galactosaminide. The activity assay of the urinary NAGase is of diagnostic importance, because its activity rises at an early stage of renal disorder. Rapid and accurate assay of an enzyme can be achieved by the technique called rate assay, in which an enzyme solution to be assayed is simply mixed with a substrate solution in an optical cell, and the change in the absorbance at an appropriate wavelength is recorded by means of a recording spectrophotometer. 4-Nitrophenyl N-acetyl-fl-D-glucosaminide (NP-GlcNAc), routinely used as an artificial substrate for the end-point assay of NAGase, has been considered not to be usable for the rate assay, because the aglycon, 4-nitrophenol, released from the substrate is colorless and optically indistinguishable from the substrate at pH near 5, where the enzyme acts optimally. The standard assay method with this substrate involves the mixing of the enzyme with the substrate solution at pH near 5, the addition of alkali to stop the reaction, and at the same time to ionize the aglycon to 4-nitrophenoxide after a definite time interval (e.g. 5 min), and the measurement of the absorbance at 400 nm [-1-4-]. The strategy to design an artificial substrate for the rate assay of NAGase has been to synthesize an N-acetyl-fl-D-glucosaminide having an aglycon which is a rather strong acid and ionizes at pH low enough for the enzyme to be active, but such a glycoside may be unstable and difficult to synthesize (e.g. [5]). NP-GlcNAc could, however, be used as an artificial substrate if the released aglycon, 4-nitrophenol, is ionized at pH near 5 by means of an additive which promotes ionization of 4-nitrophenol. Diethylaminoethyle-cyclodextrin (DE-CD) is such an additive, which has been shown to promote ionization of 4-nitrophenol to 4-nitrophenoxide at pH near or below 5 ([-6], also shown in Fig. 1). This paper presents a new rate assay method of NAGase using NP-GIcNAc and DE-CD.
2. Materials and methods 2.1. Chemicals and urine samples Human placental NAGase (6.1 mg protein/ml, 9.2 units/ml) and NPGIcNAc were products of Sigma Chemical Co. The enzyme was diluted 600-fold in 1% bovine serum albumin containing 0.9% NaC1 and stored
14. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
55
0.5[ DE
(9 (3 r--
0.3
-2
0 JO
0.2
<:
2
4
6
8
10
pH Fig. 1. Effect of DE,-CD on the coloration due to ionization of 4-nitrophenol (0.02 mmol/l) in 0.05 mol/1 glycolate buffer. Curve N, coloration (A40o) of 4-nitrophenol in the absence of any additive; curve DE, coloration (A420) of 4-nitrophenol in the presence of 0.5% DE l ~-CD.
in a refrigerator as a stock solution. DE17-CD (DE,-CD of n = 17; n is the number of diethylaminoethyl groups introduced to ~CD) was prepared as described in [6]. Several urine samples from patients with various kinds of renal disorder (nephrosis, interstitial nephritis, diabetic nephrosis, etc.) were obtained from a hospital in Tokyo.
2.2. Standard assay method of NAGase The assay method in I-3] was used as the standard. The reaction mixture contained 2.4 mmol/l NP-GlcNAc in 0.1 mol/l citrate buffer, p H 4.5. The stock solution was preincubated at 37°C and mixed with the enzyme solution of appropriate dilution at time zero. The volume of the reaction mixture was 3.0 ml. After 5 rain of incubation, 1.0 ml of 1 mol/1 sodium carbonate was added to the reaction mixture to make the pH 10.2, and the concentration of the aglycon released was estimated by the absorbance at 400 n m (A4o0). The A4o o at time 0 (sodium carbonate was added simultaneously with the enzyme) was recorded, and subtracted from that after 5 min for every sample. The enzymatic activity was expressed in units (one unit of the enzyme releases 1 #mol of aglycon from the substrate per min).
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H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
2.3. Millimolar absorbance of 4-nitrophenoxide ion The millimolar absorbance of 4-nitrophenoxide is 18.6 at 400 nm in the absence of any cyclodextrin derivative. It changes little in the presence of DE,-CD of n smaller than 10. However, it changes significantly in the presence of DEIT-CD, and is 16.8 at 400 nm and 22.0 at 420 nm [6]. 3. Results
3.1. Choice of buffer solution for the rate assay In order to find a suitable buffer for the rate assay, the effect of the buffer solution to the enzymatic activity was estimated by the standard assay system, in which citrate (recommended in [3]) was replaced with acetate, glycolate, or succinate of the same pH. Table 1 illustrates that the enzyme acts poorly in acetate buffer, but acts well in citrate, succinate, or glycolate. Since the citrate and succinate are known to suppress the DEn-CDassisted ionization of 4-nitrophenol to 4-nitrophenoxide [6], they cannot be used for the rate assay with NP-GlcNAc as a substrate, in which the released 4-nitrophenol is quantitated by automatic spectrophotometry without interrupting the enzymatic reaction by the addition of alkali. The effect of glycolate on the DEn-CD-assisted ionization of 4-nitrophenol was, thus, tested by the method detailed in our previous paper [6], and the results are shown in Fig. 1. The pKa of 4-nitrophenol in 0.05 mol/1 glycolate buffer was estimated to be 5.40 in the presence of 0.5% DEIT-CD, whereas that in 0.1 mol/1 acetate buffer was 5.23 [6], i.e. DEI7-CD exerts nearly its full effect to lower pKa of 4-nitrophenol in 0.05 mol/l glycolate buffer. The pKa of 4-nitrophenol is 7.14 in the absence of any cyclodextrin derivatives (also shown in Fig. 1).
Table 1 Comparison of NAGase activity in various buffer solutions Buffer (pH 5.0)
Activity (nmol/min)
Relative activity (1.00 for citrate)
Acetate Glycolate Succinate Citrate
5.44 9.77 8.95 8.22
0.66 1.19 1.09 1.00
The enzymaticactivity was measured by the standard method except for the buffer used. See text for details.
1-1.Shibata, T. YagiI ClinicaChimicaActa 251 (1996) 53-64
57
3.2. Effect o f DEn-CD on the enzymatic activity As DE,-CD will be contained in the enzymatic reaction mixture to increase the ionization of 4-nitrophenol, the effect of some DE,-CDs on the enzymatic activity was estimated by the standard assay method. Table 2 indicates that DEn-CD of n smaller than 5 exerts no effect on the enzymatic activity, but DE17-CD was slightly inhibitory, and its effect must be calibrated in the rate assay. This table also gives the pKa values of 4-nitrophenol in 0.5% of various DE,-CDs. 3.3. ,Rate assay o f NAGase with NP-GIcNAc The reaction mixture containing 7.2 #mol NP-GIcNAc, 15 mg DE17CD, and 0.15 mmol glycolate buffer, pH 5.0, in 2.9 ml was placed in an optical cell (optical path; 10 mm) installed in a recording spectrophotometer, and preincubated for 5 min at 37°C. Then a 0.1-ml portion of a properly diluted NAGase solution was added to the mixture, and A ~ moniitored. The results are shown in Fig. 2. It is clearly shown that A ~ increases linearly with time, and the rate of A40o increase (AA40o/min) is proportional to the amount of NAGase added to the reaction mixture (the insert in the figure). It has been confirmed that A400 does not change at all under similar experimental conditions in the absence of any cyclodextrin derivatives (not shown).
3. 4. The apparent pH optimum and the real pH optimum The rate assay described in the preceding section was repeated except that the amount of NAGase was fixed, and the pH of the reaction mixture was changed. The results are shown in Fig. 3. Apparently the rate of A400 Table 2 Effect of DEn-CD on the activity of NAGase and on pKa of 4-nitrophenol Additive (0.5°/.)
Relative activity (1.00 for non-addition)
pK, of 4-nitrophenol
None DEo-CD DEs-CD DEIT-CD
1.00 1.00 1.02 0.70
7.14 6.32 5.78 5.40
The enzymatic activity was measured by the standard method, except that cyclodextrin derivative was contained in the reaction mixture as indicated, and the buffer(pH 5.0) used was 13.05mol/l glycolate.DEo-CD is non-substituted a-cyclodextrin.
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H. Shibata, T. Yagi / Clinica Chimica Acta 251 (1996) 53-64
A 400 A:4OO.]min. × 100 0.14
0.12
0.10 |
-40
-20
!
0 Time
20
40
(s)
Fig. 2. Relationship between the rate of A4oo increase and the amount of NAGase contained in the reaction mixture. A mixture (2.9 ml) of 2.48 mmol/] NP-GlcNAc and 0.517% DE,v-CD in 0.0517 mol/l glycolate buffer, pH 5.0, was placed in an optical cell (optical path: l0 ram) in a recording spcctrophotometer at 37°C. An enzyme solution (0.1 ml) was added at the arrow, and the change in ,4400 was monitored. The enzyme added was (from the bottom of the figure) 0.00, 2.87, 5.74, 8.61, or 11.48 milliunits/ml of the reaction mixture. The inset indicates the relationship between AA4oo/min and the amount of the enzyme (milliunits/ml) contained in the reaction mixture.
increase is maximum at pH 5.7 (open circles). However, the actual reaction rates expressed in nmol/min (milliunits) must be calculated using the degrees of ionization of 4-nitrophenol at different pH values calculated from the pKa of 4-nitrophenol under the experimental conditions, as follows. degree of ionization = 1/{ 1 + exp[2.3026(pK,- pH)]} where pKa is 5.40 (see above). The pH-activity relationship thus calculated is shown in Fig. 3 (solid circles). The real pH optimum for NAGase is 4.8.
3.5. Kinetic parameters The rate assay of NAGase was repeated with the amount of NAGase
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
0 •
59
AA400/mill x 100 nmol/rain
// •
44
I
I
I
5
6
6.8
pH
Fig. 3. The pH- reaction rate relationship of NAGase. The reaction mixture contained 2.4 mmol/l NP-GIcNAc, 6.0 milliunits/ml of NAGase, and 0.5% DE17-CD in 0.05 mol/l glycolate buffer at pH between 4.58 and 6.70. Ordinate: the apparent reaction rate expressed in terms of AA~0o/min x 100 (O) and the actual rate of aglycon release expressed in terms of nmol/min per ml of the reaction mixture (.).
fixed and the pH of the reaction mixture at 5.50 (at which the ionization degree is 0.557), but the substrate concentration was varied. The results are shown in Fig. 4. From the Lineweaver-Burk plot (inset of the figure), the Km of NAGase for NP-GlcNAc was estimated to be 0.35 mmol/1, a value consistent with those reported for NAGases from other animal origins I-3,7,8]. This indicates that DE17-CD, although slightly inhibitory for the enzyme, does not have much interference for the binding of the enzyme to the substrate. 3.6. Comparison of the rate assay with the standard assay method The NAGase activities of urine samples from patients of various degrees of renal disorder were measured by the rate assay developed in this
60
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
V-I
.
.~
.
.
.
2
4
I-
1 2 substrate concentration
'S ol
s (raM)
Fig. 4. The relationship between the reaction rate (v, in nmol/min per ml of the reaction mixture) and the concentration of NP-GIcNAc (s, in retool/l). The reaction mixture contained 4.7 milliunits/ml of NAGase, 0.5% D E I 7 - C D , and varying concentrations of NP-GlcNAc in 0.05 mol/l glycolate buffer, pH 5.50, at which the degree of ionization of 4-nitrophenol is 0.557. The inset is the Lineweaver-Burkplot.
research and by the standard assay m e t h o d [3] at the same time, and the activities of both methods are compared as shown in Fig. 5. The correlation coefficient r between the data from these two methods was 0.991.
3. 7. Reliability and reproducibility o f the rate assay method Each of two urine samples, one from a nephritic patient and the other from a healthy individual, was divided into 5 portions and stored frozen at - 8 0 ° C . At intervals, the frozen samples were taken out, and the NAGase activities were determined by the rate assay method. The results are given in Table 3.
4. Discussion For the routine assay of enzymatic activities of biological fluids, the advantage of the rate assay over the end-point assay based on either spectrophotometry or spectrofluorometry is obvious. In the end-point assay, the operator has to measure the absorbance or fluorescence at least twice, at time zero and after a definite time interval for every sample,
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
61
0.06
0.04 .c_
o
o
'~
o
0.02
°o° o
o
o
0.00
,
i 2
,
, 4
,
, 6
, 8
n m o l / m i n p e r m l of t h e r e a c t i o n m i x t u r e
Fig. 5. Relationship between the NAGase activities measured by the rate assay method and those assayed by the standard method. The rate assay was conducted with 2.4 mmol/I NP-GIcNAc and 0.5% DE17-CD in 0.05 mol/l glycolate buffer, pH 5.5, at 37°C, and the observed rate of A42o increase (AA42o/min) was plotted (ordinate) against the enzymatic activity (nmol/min per mi of the reaction mixture) measured by the standard assay (abscissa). The number of samples was 18:15 from patients of renal disorder, 1 from a healthy individual, 1 (e located upper right) from human placenta (product of Sigma Chemical Co.), and 1 (e near the origin) for the control without enzyme. The correlation coefficiient r was 0.991.
because biological fluids such as urines or sera may be colored differently, and may contain fluorescent or fluorescence-quenching substances. A simple and reliable rate assay method is desired to meet an increasing demand for the activity assay of enzymes of diagnostic importance. Urinary NAGase is one of the diagnostic enzymes that increases at an early stage of various kinds of renal disorder. Several artificial substrates are proposed for the assay of this enzyme. NP-GIcNAc is one of the most prevalent substrates for the end-point assay of this enzyme: this substrate is easy to synthesize and commercially available, soluble in water, and its aqueous solution can be stored without decomposition. It is, however, difficult to conduct a rate assay with this substrate, because the aglycon 4-nitrophenol released from this substrate is rather a weak acid, and does not ionize to be spectrally distinguishable from the substrate at low pH, where: the enzyme acts optimally. Restricting our discussion to the artificial substrates for NAGase to be of N-acetyl-fl-D-glucosaminides of substituted phenols, rate assay may be
62
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
Table 3 Reproducibility of the rate assay method applied to urine samples Storage after sampling (days)
2 6 9 13 16
No. of measurements n
NAGase activity (AA420/min) Urine from a nephritic patient
Urine from a healthy individual
Activity
a
Activity
a
10 10 10 10 10
0.04778 0.04845 0.04828 0.04834 0.04798
0.00065 0.00093 0.00091 0.00106 0.00095
0.00735 0.00736 0.00714 0.00714 0.00752
0.00033 0.00032 0.00026 0.00011 0.00028
50
0.04817
0.00095
0.00730
0.00031
The rate assay was conducted with 2.4 mmol/1 NP-GIcNAc and 0.5% DE17-CD in 0.05 mol/l glycolate buffer, pH 5.5, at 37°C, and the rate of A42o increase (AA42o/min) was measured in an optical cell (optical path: 10 mm). Each reaction mixture contained 0.1 volume of the urine sample. Using Fig. 5, the observed rate of A,2 o increase (AA,2o/ min --- 0.04817) with the urine sample from the nephritic patient was converted to the reaction rate of 6.67 nmol/min per ml of the reaction mixture, i.e. 66.7 nmol/min per ml of the urine sample. Similarly, the NAGase activity of the urine sample from the healthy individual was estimated to be 7.9 nmol/min per ml of the urine sample.
possible if the aglycon of the substrate is rather a strong acid and ionizes upon enzymatic release at pH near 5. Such artificial substrates proposed for practical uses include 2-chloro-4-nitrophenyl fl-GlcNAc (I) [9-1, 3,4dinitrophenyl fl-GlcNAc (II) [10], 2-fluoro-4-nitrophenyl ~-GlcNAc (III) [11], etc. The pKa of the aglycons from substrates I and II are 5.45 and 5.38, respectively. A disadvantage of I is its low solubility: the reaction mixture containing this substrate must be supplemented with complicated additives to improve its solubility for use in the rate assay [9]. II is much more soluble, but slowly decomposes in solution upon storage at 4°C. It is recommended to use its stock solution within a week [10]. A disadvantage of Ill is its rather high Km. The rate assay method presented in this paper solves these problems. NP-GlcNAc is commercially available, sufficiently soluble, and its solution can be stored in a refrigerator for a year without appreciable decomposition. The aglycon released from NP-GlcNAc is, by itself, a weak acid (hence its glycoside is chemically stable), but behaves like a stronger acid to develop a yellow color at lower pH in the presence of DEI7-CD, whose
H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
63
effect on NAGase is marginal. Synthesis of D E , - C D of n near 17 may be laborious [6], but does not require any special technique of organic chemistry. It is advisable to synthesize DE,-CD of n near 17 in bulk to evaluate experimentally its ability of lowering pKa of 4-nitrophenol and its effect on the activity of NAGase under the assay conditions before its use in the rate assay of NAGase. The new rate assay method proposed in this research was shown to be reliable and reproducible (Table 3), and gave comparable results to those obtained by the standard assay method as shown in Fig. 5. This research was conducted with DE17-CD which had been prepared in bulk, but DEn-CD of n < 17 may be similarly employed. As every DE,-CD preparation with different n may promote ionization of 4-nitrophenol differently I-6], its effect on lowering pKa of 4-nitrophenol in the reaction mixture must be estimated for every lot of preparation. For example, DEs-CD will be used as an additive for the rate assay, but the sensitivity with this additive may be a little lower, because the pK a of 4-nitrophenol with this additive is 5.78 (Table 2), and hence the degree of ionization is calculated to be 0.345 at pH 5.5. A possible recipe for the rate assay of NAGase is as follows. Prepare a stock solution containing 4.8 mmol/l NP-GlcNAc and 1% DE17-CD in 0.1 mol/1 glycolate buffer, pH 5.50, a non-inhibitory aseptic being added if neces,;ary. Introduce the stock solution and a properly diluted sample (urine or other body fluid) to a reaction cell placed in a spectrophotometer at a ratio of 1:1, and monitor A400 or A420. The ratio of the stock solution and tile urine sample may be variable as far as the composition of the final mixture is kept unchanged. The reaction rate (enzymatic activity) can be conveniently read directly from calibration plots as shown in Fig. 5 (such a figure must be prepared for a given lot of DE.-CD sample), or can be calculated from the rate of the absorbance increase (AA400/min or AA420/ min, ionization degree of 4-nitrophenol at pH 5.50, and the millimolar absorbance coefficient of 4-nitrophenoxide in the presence of 0.5% DE17CD (16.8 at 400 nm or 22.0 at 420 nm).
References
[1] Li YT, Li SC. ~t-Mannosidase,fl-N-acetyl-hexosaminidase,and fl-galactosidasefrom jack bean meal. Methods Enzymol 1972;28:702-713. [2] AgrawalKM L, Bahl OP. a-Galactosidase,fl-galactosidase,fl-glucosidase,fl-N-acetyigilucosaminidase, and ~t-mannosidase from pinto beans. Methods Enzymol 1972;28:720-728. [31 Tarentino AL, Maley F. fl-N-Acetylglucosaminidasefrom hen oviduct. Methods Enzymoi 1972;28:772-776.
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H. Shibata, T. Yagi I Clinica Chimica Acta 251 (1996) 53-64
[4] McGuire EJ, Chipowsky S, Roseman S. fl-N-Acetylglucosaminidase, ~t-N-acetylgalactosaminidase, and fl- galactosidase from Clostridium perfringens. Methods Enzymol 1972;28:755-763. I'5] Jones CS, Kosman DJ. Purification, properties, kinetics, and mechanism of E-Nacetylglucosaminidase from Aspergillus niger. J Biol Chem 1980;255:11861-11 869. 1'6] Yagi T, Aoshima R, Kuwahara M, Shibata H. Preferential inclusion of nitrophenoxide ion over its conjugate acid in polycationic cyclodextrin: pK, decrease of nitrophenols by host-guest interaction with diethylaminoethyl-cyclodextrins. J Inclusion Phenom 1993;16:231-243. [7] Frohwein YZ, Gatt S. Isolation of fl-N-acetylhexosaminidase, fl-N-acetylglucosaminidase, and fl-N-acetylgalactosaminidase from calf brain. Biochemistry 1967; 6:2775-2782. 1-8] Geiger B, Calef E, Arnon R. Biochemical and immunochemical characterization of hexosaminidase P. Biochemistry 1978;17:1713-1717. 1-9] Makise J, Ichikawa K, Yoshida K, Watanabe S. New derivatives of glucosaminide and the assay reagents using these derivatives as substrates for N-acetyl-fl-oglucosaminidase. Patent 1742610, Japan, 1993. [I0-1 Yagi T, Hisada R, Shibata H. 3,4-Dinitrophenyl N-acetyl-fl-o-glucosaminide, a synthetic substrate for direct spectrophotometric assay of N-acetyl-fl-o-glucosaminidase or N-acetyl-fl-D-hexosamirndase. Anal Biochem 1989;183:245-249. [111 Kurihara T, Kamimura M, Hayashi Y, Teshima S. Derivatives of N-acetyl-fl-oglucosaminide and the assay reagents using these derivatives as substrates for Nacetyl-fl-D-hexosaminidase. Patent 1847729, Japan, 1994.