Electrophoretically mediated micro reaction


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Electropholetically mediated reaction of glycosidase: An automated kinetic analysis at a nanoliter scale

Yoshimi Kanie and Osamu Kanie

Mitsubishi Kagaku Institute of Life Sciences (MITILS)


Oligosaccharides existing on the cell surface and in the extracellular matrix as a component of glycoproteins and glycolipids are involved in a variety of biological phenomena. Methodological investigation directed toward revealing the functions of oligosaccharides is, therefore, of extreme importance. We have been studying carbohydrate related enzymes, especially oligosaccharide processing enzymes, and reported usefulness of capillary zone electrophoresis (CZE), where analysis was carried out separately after the reaction. However, further down scaling of enzyme reaction is necessary for some cases. Rec
ently, an approach in which the capillary is used not only as an isolation field but also as a reaction vessel where the enzymatic reactions take place is recognized to be highly valuable. This electrophoretically mediated microanalysis (EMMA) is advantageous because the amounts of enzyme and substrate can be minimized. The practical investigation of EMMA is very important in connection with currently focused chip-based technologies.

There are basically two ways to mix two components in a capillary under electrophoretic conditions, the continuous format and the plug-plug format. In contrast to the use of a capillary filled with one of the reactants in the continuous format, the plug-plug format is based on a plug-plug interaction. When two compounds forming individual plugs have different electrolytic mobilities, the reaction proceeds while one of the components is passing the other. The reaction product is then resolved based on the individual electromobilities in the electric field and passed through the detector along the way to the end of the capillary. However, because less sample is consumed and the reaction time is shorter, it becomes less sensitive compared to the continuous method. Despite this problem, the method has been successfully applied to obtain the Michaelis constant (Km) in various enzyme reactions.

In this study, we present the examples of EMMA of glyco-enzyme using a plug-plug format. Here, it was essential to overcome the problem of the incompatibility of the conditions for the enzyme reaction and carbohydrate analysis. For the analysis of carbohydrates, borate buffer is commonly used at alkaline pH to introduce negatively charged functionality based on the formation of a borate complex of hydroxyl groups. However, it was found that enzyme reaction did not proceed under these conditions.


Model reaction

See Figure 1.


Enzymes; β-Glucosidase (β-Glc-ase, EC from Sweet almond, α-glucosidase (α-Glc-ase, EC from Saccharomyces sp., β-galactosidase (β-Gal-ase, EC from Aspergillus oryzae, β-N-acetylglucosaminidase (β-GlcNAc-ase, EC from bovine kidney, α-mannosidase (α-Man-ase, EC from jack bean, α-fucosidase (α-Fuc-ase, EC from bovine kidney

Substrates; p-Nitrophenyl-β-glucoside (PNP-β-Glc), p-nitrophenyl-α-glucoside (PNP-α-Glc), p-nitrophenyl-β-galactoside (PNP-β-Gal), p-nitrophenyl-β-N-acetylglucosaminide (PNP-β-GlcNAc), p-nitrophenyl-α-mannoside (PNP-α-Man), p-nitrophenyl-α-fucoside (PNP-α-Fuc)

Internal standard; Uridine

CE instrument: Beckman P/ACE System 5500

CE condition

Capillary: 75 mm (i.d.) x 37 cm total length, 30.5 cm to detector (bare fused-silica)

Capillary temperature: 37°C

Separation buffer: 40 mM Sodium borate buffer, pH 9.2

Field strength: 18 kV

Detection: PDA UV detector

Sample introduction mode: pressure

Sample introduction process

1) Washing with 0.1 N NaOH (2 min), [and regenerated with water (1 min)]

2) Equilibration of the electrolyte buffer (2 min)

3) Injection of a solution containing enzyme in phosphate buffer (3 sec)

4) Introduction of phosphate buffer (1 sec)

5) Injection of a solution of substrate in phosphate buffer (5 sec)

Concentration and pH of phosphate buffer

See Table 1

The stock solutions of enzyme, substrate and buffers used in the EMMA were kept at 20°C on reservoir.

Estimation amounts

Poseuille equation

V = ΔPπr4t/8ηL

ΔP: the pressure drop across the capillary during injection, r: capillary radius, t: injection time,η: the viscosity of the buffer, L: total length of the capillary from inlet to outlet.

The length and volume (V) of the enzyme and substrate plugs are estimated to be 6.5 and 10 mm and 29 and 45 nL, respectively, which were isolated by a 2 mm (9 nL) phosphate buffer plug.


A micro reaction in capillary under electrophoretic conditions is schematically presented in Figure 2A. The isolated plugs of enzyme and substrate are introduced into the capillary. The order of the introduction is determined based on the relative electromobility. The reaction proceeds during plug-plug interaction (t2-t1), and the individual plugs of substrate, enzyme and products are separated. Figure 2B shows an imaginative electropherogram of the reaction recorded at ld. Figure 2C shows the illustration of the capillary inside at tx.

We concluded the substrate buffer concentration should be under 200 mM after examination of the effect of the phosphate buffer concentration on the sensitivity of PNP-ol because we observed a disturbance of the baseline, which is probably associated with the front and end boundaries of the phosphate buffer, which affected the substrate and PNP-ol peaks, respectively. The effect of the pH was then investigated. The optimal transformation was observed around optimal pH of each enzyme.

Using optimized conditions for the kinetic analysis of each enzymes for micro reaction, we obtained the kinetic parameters of the reaction. Figure 3 shows the electropherograms of each reaction. The assay was performed at 37°C and detected at 214 nm. The Km values were obtained by a double-reciprocal plot where the relative area was used to indicate product concentrations, which was used instead of the initial velocity of the enzyme reaction. Figure 4 represent the example of the double-reciprocal plot for α-Glc-ase reaction. The enzyme reaction is described by the Michaelis-Menten equation,

v = Vmax[S]/(Km+[S])

where v is the initial rate of the given reaction, [S] is the substrate concentration, and Km is Michaelis constant. Although, the reaction time is not directly given in EMMA because of the heterogeneous nature of the reaction that takes place during the plug-plug interaction, linearity was obtained for the relative area of the product PNP-ol over uridine during the experiment at a fixed concentration of the phosphate buffer. Therefore, we used the area as an equivalent value of v to obtain the constant. The Michaelis constants thus obtained were shown on the Table 2 together with those obtained by conventional method.

In further examples to show EMMA's potential utility in the functional analysis of carbohydrate related enzymes and/or carbohydrates were shown in Figure 5 and 6. Figure 5 shows the electropherograms of the selective reaction which used a mixture of PNP-β-glycosides of glucose and galactose with β-Glc-ase. The structural difference of these glycoside is a single chirality at C-4 position of sugar moiety. It was clearly shown in Figure 5A, that these compounds were resolved under the condition due to the presence of borate buffer. Furthermore, in Figure 5B, it was an evident that the β-Glc-ase catalyzed reaction proceeded where the formation of PNP-ol was observed and the area of PNP-β-Glc was decreased while PNP-β-Gal was unaffected. Figure 6 shows the electropherograms of the reaction using two enzymes and corresponding substrates (PNP-β-Glc and ONP-β-Gal) for the potential use of a mixture of functi
onally and/or structurally related enzymes. To confirm both reactions, p-nitro- and o-nitrophenyl glycosides were used. Figure 6A and B show the electropherograms, that two substrates and phenols were resolved well under EMMA condition without enzymes, respectively. Figure 6C shows that the mixed enzymatic reaction is possible, and corresponding product peaks could be easily identified by PDA detection as shown in the box.


The native glycoenzyme related reactions using a plug-plug format under EMMA conditions were achieved after investigation of the detailed reaction conditions. An evaluation of the kinetic constant was also carried out under the condition. As a result, the scale of the enzymatic reaction was radically reduced (at least a 1000 fold improvement compared to off-line analysis of the reaction carried out in a microcentrifuge tube). Thus, we were able to perform the enzymatic reaction at a nanoliter scale, whereas the limit in reaction volume for traditional methods is µL scale due to surface tension problems in our hands. As a representative of this micro reaction under EMMA conditions, glycoenzyme reaction were performed and Km values similar to those reported were obtained. This process was carried out automatically using a temperature-controlled auto-sampler in order to eliminate routine handling and to speed-up the process. Furthermore, we envisioned that it will be important to analyze a small amount of enzyme mixture for their activities and specificities, and showed it is possible based on the microreaction under electrophoresis.


Kanie, Y.; Kanie, O. "Electrophoretically Mediated Microscale Reaction of Glycosidase: Kinetic analysis of some glycosidases at nanoliter scale", Carbohydr. Res., 2002, 337, 1757-1762.

Kanie, Y.; Kanie, O. "Electrophoretically Mediated Reaction of Glycosidase at a Nanoliter Scale", Electrophoresis, 2003, 24, 1111-1118.