A pH-sensitive assay for galactosyltransferase

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ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 330 (2004) 219–226 www.elsevier.com/locate/yabio

A pH-sensitive assay for galactosyltransferase Chenghua Deng and Rachel R. Chen* Chemical Engineering Department, Virginia Commonwealth University, 601 W. Main Street, Richmond, VA 23284, USA Received 9 December 2003 Available online 20 May 2004

Abstract We report here a new pH-indicator-based assay for galactosyltransferase. The method is simple and fast, requires no specialized equipment, labeled substrate, or other expensive materials, and is thus expected to have broad applications including automated high-throughput screening. The method is based upon the detection of absorbance change of a pH indicator, phenol red, in response to proton release that accompanies the galactosyltransferase-catalyzed galactose transfer. The assay was used to compare three galactosyltransferases in our collection. As demonstrated here, subtle differences in substrate specificity were readily discerned with this sensitive method. All three enzymes accept both N -acetylglucosamine and glucose as acceptor but the relative activity varies with the origin of the enzyme. The method was demonstrated to be useful in the initial characterization of recombinant galactosyltransferase from crude cell extract. Optimal metal cofactor Mn2þ concentration and temperature were determined with the method. Overall, the method offers a great improvement over current methods in reducing time and material consumption. It is the first pH-sensitive method for galactosyltransferase. The principles of using pH indicator in galactosyltransferase assay should be applicable to other glycosyltransferase enzymes. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Galactosyltransferase; Proton release; Phenol red; pH-sensitive assay; pH indicator

Galactosyltransferases are a family of enzymes catalyzing galactose transfer from a donor substrate UDP-galactose to an acceptor molecule. Specifically, b-1,4-galactosyltransferase (GalT1)1 catalyzes the transfer of galactose from UDP-galactose to N -acetylglucosamine (Scheme 1). The disaccharide product of the reaction, N -acetyllactosamine, is the core structure of glycans associated with many important biological processes and human diseases. For example, it occurs in sialyl Lewisx , an oligosaccharide associated with inflammation processes, and in Gal(1,3)bGal(1,4)bGlcNAc associated with hyperacute rejection of xenografts [1]. The biomedical relevance of the enzyme has, in

*

Corresponding author. Fax: 1-804-828-3846. E-mail address: [email protected] (R.R. Chen). 1 Abbreviations used: UDP, uridine diphosphate; GlcNAc, N -acetyl glucosamine; Glc, glucose; Gal, galactose; NAD, nicotinamide adenine dinucleotide; HPLC, high-performance liquid chromatography; GalT, b-1,4-galactosyltransferase (from bovine milk); IPTG, isopropyl thiogalactopyranoside; OD, optical density. 0003-2697/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.03.014

recent years, stimulated research on this enzyme and its associated glycans [2–5]. A reliable, sensitive assay of this enzyme is essential to all these activities. Palcic and Sujino [6] thoroughly reviewed current assay methods for glycosyltransferases. Radiochemical assays are the most frequently used due to their high sensitivity. Immunological methods [7] are also sensitive and are able to identify reaction products, but they require very specific antibodies or lectins, which are expensive or, worse, not readily available in most cases. A spectrophotometric assay developed in 1970s requires two additional enzymes and is based on detection of reaction product UDP (Scheme 1) by coupling the glycosyltransferase-catalyzed reaction with two other enzymatic reactions that involves NADH/NAD cofactors [8,9]. Various chromatographic methods developed for glycosyltransferase assay can combine product identification with enzyme assay, but they require substrate fluorescence labeling or special detection methods [10]. Mass-spectrometry-based assays [11] are powerful with regard to speed and accuracy but are not widely used

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Scheme 1. Reaction catalyzed by GalT1.

due to the high cost of the instrument. These methods and other more recent modifications [12], requiring substrate labeling, high instrument investment, or additional enzymes or antibodies, are all based upon the detection of the substrate consumption of the formation of oligosaccharide product or nucleotide product. Interestingly, proton release that accompanies the glycosyltransferasecatalyzed reactions has never been exploited in the enzyme assay. This is surprising as pH change due to proton release of an enzymatic reaction is a feature that can be conveniently used in assay development, and indeed it is used for assays for a wide range of enzymes including kinase [13], lipase [14], and phospholipase [15]. In our research focusing on biocatalytic synthesis of complex carbohydrates, we developed an HPLC method based on ion-exchange chromatography and electrochemical detection [16]. It afforded reliable assay but was very time consuming. In developing various enzymatic synthesis strategies including comparing various glycosyltransferases and investigating numerous biocatalytic conditions, we realized that we need a much faster assay. Therefore, we undertook this study to develop a pH-sensitive assay for b-1,4-galactosyltransferase and to illustrate the methodology that is expected to be applicable to other galactosyltransferases and other glycosyltransferases.

Materials and methods

drate column PA-10 (250  4 mm) was used in the analysis and 40 mM NaOH was used to elute the product followed by a regeneration of the anion exchange column with 200 mM NaOH at the end of each run. A typical run takes about 45 min. Enzyme preparation Two recombinant enzymes were expressed in Escherichia coli, the genes were originated from Neisserria meningitidis and Helicobacter pylori and designated LgtB [17] and HP0826 [18], respectively. Both enzyme constructs were gifts from Dr. Wakarchuk of the National Research Council of Canada. The crude enzymes (LgtB and HP0826) were prepared according to the procedure of Blixt et al. [3] with some modifications. A glycerol stock of E. coli strains carrying appropriate plasmid was inoculated into LB (10 ll stock/ml LB) with 150 lg/ml ampicillin and incubated at 37 °C overnight. Then 1 ml of the overnight culture was transferred into 100 ml LB with 150 lg/ml ampicillin and incubated at 37 °C until the OD600 reached between 0.6 and 0.8 (2–2.5 h). Protein expression was induced by adding IPTG to a final concentration of 0.4 mM and the culture was further incubated for 6 h. Cells were harvested by centrifugation. The pellet was resuspended in 1 ml phosphate buffer (2 mM, pH 8.0). Enzymes were released by sonication. The pellet was removed by centrifugation, and supernatant (containing crude enzyme) was collected and stored at )20 °C or used as indicated in this study.

Materials and equipment All the reagents including sodium phosphate (monobasic and di-basic), phenol red, N -acetylglucosamine, UDP-galactose, UDP-glucose, galactose, glucosamine, lactose, glucose, N -acetylgalactosamine, isopropyl thiogalactopyranoside (IPTG), and a-lactalbumin are from Sigma. Galactosyltransferase from bovine milk is also from Sigma. The absorbance at 557 nm of phenol red was recorded with a Perkin–Elmer Lambda 40 UV–Vis spectrometer.

Calibration curve: relationship between proton production and absorbance of the pH indicator In a 2 mM sodium phosphate buffer (pH 8, 1 ml) containing 0.01 mM phenol red, 0.1 mM MnCl2 , 10 mM N -acetylglucosamine, and LgtB or HP0826 (50 ll), different amounts of hydrochloric acid (10 mM) were added to final concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mM, and the OD557 was recorded. A quantitative linear relationship between proton concentration and absorbance was established.

HPLC method Enzyme assay Lactose synthesis catalyzed by GalT was detected by a high-pH anion exchange chromatography system with pulsed amperometric detection (Dionex). A carbohy-

For this assay, 0.01 mM phenol red, 0.1 mM MnCl2 , 10 mM N -acetylglucosamine, and an enzyme of appro-

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priate volume (typically 50 ll of LgtB, or 50 ll of HP0826, or 20 ll of GalT) were mixed with phosphate buffer (2 mM, pH 8). The reaction was started by adding UDP-galactose to a final concentration of 2 mM, and the absorbance at 557 nm was recorded for each sample at 10-s intervals for a total of 5 min. The total volume is 1 ml. All the measurements were carried out at 30 °C except as indicated otherwise. The activities of enzymes were calculated from the calibration curve. To compare substrate specificity of the three enzymes, additional acceptor substrates including glucose, galactose, lactose, N -acetylgalactosamine, and glucosamine were used as acceptor. For donor substrate specificity, UDP-glucose and UDP-galactose were used, and N -acetylglucosamine was used as acceptor. All measurements were done in triplicate and values averaged. For reactions catalyzed by GalT, 0.2 mg/ml a-lactalbumin was added except as indicated otherwise. Effect of temperature on the activity of enzyme Optimal temperatures were determined under conditions as described above, except that measurements were taken at different temperatures (20, 30, or 37 °C). Effect of a-lactalbumin on enzyme activity Influence of a-lactalbumin on enzyme kinetics was determined under essentially the same conditions as described above, except that a-lactalbumin was added to the mixture to a final concentration of 0.2 mg/ml. Effect of manganese on the activity of galactosyltransferase Optimal cofactor concentration was determined under essentially the same conditions as described above, except that the final concentration of MnCl2 was changed to 0, 0.05, 0.2, or 0.5 mM.

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choice of pH buffer concentration. A higher buffer concentration is not desirable as it renders the method insensitive to pH change, the basis of the detection. Buffer concentration too low is problematic as absorption of CO2 from air will cause fluctuations in pH or proton measurement. Generally, pH buffer concentration in a pH-indicator-based assay should be kept as low as possible. After an initial investigation, a buffer concentration of 2 mM was chosen. Earlier studies [13] show that a pH buffer with a pKa as close to the pKa of the indicator as possible gives the best result. For this reason, we chose phosphate buffer (pKa 7.2) in our assay development, which was found to be satisfactory. A galactosyltransferase-catalyzed reaction is a proton-producing reaction; thus pH will decrease as reaction proceeds. If the pH falls within the color-change range, the color of phenol red changes from red to yellow, with a corresponding absorbance decrease. The absorbance decrease, correlated with the proton concentration increase by an appropriate calibration curve, can then be used as the basis for quantitative analysis of galactosyltransferase. Titration using HCl was used to establish the relationship between proton concentration and absorbance of the indicator (details are described under Materials and methods). As seen in Fig. 1, absorbance measured at 557 nm is linearly correlated with proton concentration from 0 to 0.8 mM, which covers the range of proton concentration of all experiments in this study. Note that the calibration curve was generated with essentially the same enzyme mixture as that used in the actual assay omitting only the donor substrate. A typical time trajectory of absorbance change corresponding to LgtB-catalyzed proton release is shown in Fig. 2. Using the calibration curve shown in Fig. 1, the proton concentration change corresponding to the absorbance change was calculated and plotted as a

Results and discussion Method development When choosing an appropriate pH indicator to use in the assay development, we considered the pH optimal of galactosyltransferase, which is close to 8.0, and the pH range for the color change of an indicator. We chose phenol red since its pKa is 7.4 and it transits from red to yellow with associated absorbance change within a pH range of 8.4–6.8, coinciding with a pH range within which galactosyltransferase is expected to be most active, though not necessarily at its optimal. Phenol red has a peak absorbance at 557 nm; this wavelength is used in all of our subsequent experiments. Another important consideration in assay development is the

Fig. 1. Calibration curve: relationship between proton concentration and absorbance at 557 nm. The mixture contains 2 mM phosphate, pH 8.0, 0.01 mM phenol red, 0.1 mM MnCl2 , 10 mM N -acetylglucosamine, and 50 ll LgtB. OD557 was measured after addition of different amounts of hydrochloric acid (0–0.8 mM).

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Fig. 2. Change of absorbance at 557 nm with time for LgtB. The enzyme was assayed in 2 mM phosphate, pH 8.0, 0.01 mM phenol red, 0.1 mM MnCl2 , 10 mM N -acetylglucosamine, and 50 ll LgtB. The reaction was started by adding 2 mM UDP-galactose, and measurements were taken at 557 nm for 5 min with a time interval of 10 s. The measurement was repeated in triplicate and the values were averaged. The curves represent the average and standard deviation. No UDPgalactose was added in Blank.

Fig. 3. Release of proton as a function of time from an LgtB-catalyzed reaction. The enzyme was assayed in 2 mM phosphate, pH 8.0, 0.01 mM phenol red, 0.1 mM MnCl2 , 10 mM N -acetylglucosamine, and 50 ll LgtB. The reaction was started by adding 2 mM UDP-galactose, and measurements were taken at 557 nm for 2 min with a time interval of 10 s. The measurement was repeated in triplicate and the values were averaged.

function of time (Fig. 3). A linear regression was performed (R2 ¼ 0:9987) and the slope was estimated to be 0.0953. The enzyme activity was defined as the amount of enzyme required to produce 1 lmol proton per minute. The particular example (enzyme LgtB) shown in Figs. 2 and 3 was found to have activity of about 1.9 U using the following formula: 0:0953 mmol=L=min  1  103 L ¼ 1:906 lmol=min=ml 0:05 ml ¼ 1:906 U=ml:

Fig. 4. Change in absorbance as a function of time with different enzyme input. The changes in OD557 were recorded in 2 mM phosphate, pH 8.0, 0.01 mM phenol red, 0.1 mM MnCl2 , 10 mM N -acetylglucosamine, and LgtB (50, 10, 5, and 0 ll). The reactions were started by adding 2 mM UDP-galactose and measurements were taken at 557 nm for 5 min. Data were collected at each 10-s time interval. The measurement was repeated in triplicate and the values were averaged.

The detection limit of the method was estimated by varying enzyme volumes used in the assay. Fig. 4 shows the rate of the reaction decreases with the decrease of enzyme input. The linear relationship is maintained until enzyme input decreases to below 10 ll, equivalent to 0.019 U. Therefore, the detection limit for this assay with a 1 ml total assay volume is 0.019 U. If this assay is adapted for a 96-well format with a 200 ll total assay volume, the limit will be reduced to 0.0038 U/well. We compared the method with an established HPLC method in which N -acetyllactosamine, the product of galactosyltransferase-catalyzed reaction, was measured [16]. The activity measured from this assay was consistently about 50% higher than that from the HPLC method. This difference can be attributed to two main reasons. First, in this assay, proton production accompanying the glycosyltransferase-catalyzed reactions was used whereas, in the HPLC method, the disaccharide product N -acetyllactosamine was measured. The differences in chemical entities measured in the assay and different instruments used (spectrophotometer versus electrochemical detector) could be possible sources of the difference. Second, the HPLC method takes measurement at time points which are on orders of at least minutes (typically 5, 10, 20, and 30 min), whereas our proton-based assay afforded measurements every 10 s; thus the measured rate by this method reflects more closely the initial velocity (typically the first 2 min), rather than the averaged first 30 min rate in the HPLC method. We observed the proton change rate or the reaction rate decrease with time even within the first 5 min of the reaction. In Fig. 1, for example, the rate of absorption change decreased after the first 2 min, indicating that the reaction deaccelerates shortly after the initiation of the reaction possibly because of the wellknown inhibition effect of UDP. Thus it is not surprising

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to see a lower numerical value in the HPLC assay. We therefore believe that the proton-based assay is a more accurate method as it more truly reflects the initial velocity of the enzymatic reaction. In the example shown above and elsewhere in this paper, recombinant enzymes LgtB and HP0826 were

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used as cell extract without purification. Under the conditions that we used, the cell extract contains 1.36 and 1.15 mg/ml total proteins for LgtB and HP0826, respectively, of which about 20% is the recombinant enzyme as estimated from our SDS–PAGE gel analysis (data not shown). Therefore, the recombinant enzymes

Table 1 Comparison of substrate specificities for LgtB, HP0826, and GalT Donor

Acceptor

Activity (U/ml)a LgtB

HP0826

GalT

1.91  0.08

1.18  0.09

6.18  0.14

1.37  0.06

0.89  0.02

19.1  0.12

0

0

0

0

0

0

0

0

0

0

0

0.47  0.05

0.39  0.02

0

0

a The activities of LgtB, HP0826, and GalT were measured at 30 °C in 2 mM phosphate, pH 8.0, 0.01 mM phenol red, 0.1 mM MnCl2 , 10 mM N -acetylglucosamine, and 50 ll LgtB or 50 ll HP0826 or 20 ll GalT. The reactions were started by adding 2 mM UDP-galactose or UDP-glucose, and the absorbance at 557 nm was recorded for each sample at each 10-s time interval for a total of 2 min. The activity was calculated based on Figs. 1 and 2 and on the equation in the text. The measurements were done in triplicate and the data shown are the average  standard deviation.

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LgtB and HP0826 represented major components of the cell extract and the compositions of the cell extract were consistent throughout the study as identical conditions were used in each step of the enzyme preparation process. Furthermore, in all the experiments described in this paper, adequate controls were used to ensure that the proton change measured corresponds to the enzyme activity rather than to other activities in the extract. In particular, the blank contains the cell extract from the same preparation and other reagents necessary for the reaction (details under Materials and methods) except an acceptor sugar or donor substrate. As seen in Fig. 2, the change of OD of the blank in the first 2 min of the experiments was minimal compared to the sample in which UDP-Gal was added, indicating that the change of absorption and therefore proton concentration is due to the activity of the galactosyltransferase and that the interference of other components in the extract is minimal. As in many applications such as biocatalysis recombinant enzymes are used as crude extract, it is important to have a quick method for an initial characterization of enzyme properties and a quick screening method for various process conditions. The ability of this method to detect galactosyltransferase activity in a crude cell extract is therefore an advantage as it eliminates the need for purification. Substrate specificities of three different galactosyltransferases We used the method to compare three different galactosyltransferases in our collection. Of the three enzymes, two were recombinant and were expressed in E. coli. The genes were originated from N. meningitidis and H. pylori and designated LgtB and HP0826, respectively. Both enzymes catalyze galactose transfer from a donor substrate UDP-galactose to an acceptor molecule N -acetylglucosamine as shown in Scheme 1. The third enzyme is a commercial enzyme purified from bovine milk, here designated GalT, which catalyzes galactose transfer from UDP-galactose to glucose. The recombinant enzymes were prepared as described under Materials and methods. The substrate specificity of these three enzymes was investigated with a panel of six acceptors and two donors. As shown in Table 1 and Fig. 5, LgtB and HP0826 showed highest activity with donor UDP-galactose and acceptor N -acetylglucosamine, whereas the commercial enzyme GalT shows highest activity with glucose as acceptor and UDP-galactose as donor. These results are consistent with findings from the literature, indicating that our method is sensitive and useful in characterizing substrate specificity. Also consistent with earlier studies, the enzyme preparation containing LgtB was active with UDP-glucose, with about 25% activity toward UDP-

Fig. 5. Substrate specificity of LgtB. The changes in OD557 for LgtB to different substrates were recorded in 2 mM phosphate, pH 8.0, 0.01 mM phenol red, 0.1 mM MnCl2 , 10 mM N -acetylglucosamine (or glucose or glucosamine), and 50 ll LgtB. The reactions were started by adding 2 mM UDP-galactose (or UDP-glucose), and measurements were taken at 557 nm for 5 min. Data were collected at each 10-s time interval. No UDP-galactose or UDP-glucose was added in Blank.

glucose (Table 1). This is likely due to the epimerization of donor sugar as indicated by an earlier study with the LgtB construct [3] (pers. commun.). The other two enzymes show no detectable activity toward UDP-glucose. All three enzymes accept both N -acetylglucosamine and glucose as acceptor substrate. GalT also has a lowlevel activity toward disaccharide lactose, yet it shows no activity with acceptor monosaccharide galactose. As demonstrated in this example, the method is sensitive enough to differentiate closely related galactosyltransferases by their relative activities and by their subtle differences in substrate specificities. Galactosyltransferase modification by a-lactalbumin Mammalian galactosyltransferase GalT1 is exceptional in that its kinetics can be modified by regulatory protein a-lactalbumin [4,19]. The bovine commercial enzyme used in this study is also called lactose synthase. It synthesizes lactose slowly in the absence of a-lactalbumin. In the presence of a-lactalbumin, however, the reaction rate increases dramatically due to the reduced Km by the regulatory protein. The initial rate of lactose synthesis catalyzed by GalT with and without a-lactalbumin was monitored by the developed method. Fig. 6 shows the absorbance change during the first 5 min of synthesis. As illustrated in Fig. 6, in the absence of alactalbumin, the reaction barely proceeds. In contrast, rapid synthesis of lactose takes place as soon as a-lactalbumin is added to the reaction mixture. Lactose formation was monitored using an HPLC method. After 24 h, the yield of lactose synthesis based upon UDPgalactose was over 90%. As expected, a-lactalbumin has no effect on either LgtB or HP0826. When reactions

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that this sensitive method will have broad applications such as screening of enzyme inhibitors or effectors. Temperature optimal

Fig. 6. Comparison of the activities of GalT and LgtB with and without a-lactalbumin. The changes in OD557 for LgtB and GalT were recorded in 2 mM phosphate, pH 8.0, 0.01 mM phenol red, 0.1 mM MnCl2 , 10 mM glucose, 0 or 0.2 mg/ml a-lactalbumin, and 50 ll LgtB (20 ll GalT). The reactions were started by adding 2 mM UDP-galactose, and measurements were taken at 557 nm for 5 min. Data were collected at each 10-s time interval.

were carried out with N -acetylglucosamine, the presence of a-lactalbumin significantly decreases the reaction rate catalyzed by GalT (data not shown). a-Lactalbumin, therefore, is a very effective inhibitor of the enzyme, inhibiting the transfer of galactose to N -acetylglucosamine. The applicability of the method to the detection of small changes in enzyme kinetics caused by the action of regulatory molecules, as shown in this example, is a feature important for drug discovery and enzyme-based diagnostic assays or other bioassays. Thus we expect

Rapid characterization of enzymes with regard to their temperature optimal is essential to processes employing enzymes. We tested the ability of this method to determine temperature optimal in an expeditious manner. The assay was carried out with temperature as the only variable. We found that with this method it required only about 15 min total assay time for each enzyme for measurements taken at three different temperatures. We compared the assay time with a previous method established in our laboratory based on a Dionex ion-exchange chromatography [16]. The method is a significant improvement over the HPLC method as illustrated below. The HPLC method requires about 45 min for a single data point and a total of about 4–5 h for estimation of activity for a single temperature; so if an assay is conducted with three different temperatures, a total of 12–15 h of HPLC instrumental time is required. This represents about 20-fold difference in the time requirement. Table 2 shows that all three enzymes have an optimal temperature about 30 °C under the conditions described under Materials and methods. Manganese cofactor concentration Galactosyltransferase is known to require Mn2þ as cofactors [20]. But the optimal concentrations for various in vitro applications were not clearly established. As reported in the literature, they can vary over an

Table 2 Activities of three galactosyltransferases at different temperatures Temperature (°C) Activity (U/ml)

LgtB (UDP-Gal + GlcNAc) HP0826 (UDP-Gal + GlcNAc) Commercial GalT (UDP-Gal + Glc)

20

30

37

1.78  0.07 0.89  0.03 0.18  0.05

1.91  0.08 1.18  0.09 19.1  0.12

1.54  0.13 1.05  0.06 12.0  0.10

The activities of LgtB, HP0826, and GalT were measured in 2 mM phosphate, pH 8.0, 0.01 mM phenol red, 0.1 mM MnCl2 , 10 mM N -acetylglucosamine for LgtB or HP0826 (or 10 mM glucose for GalT), and 50 ll LgtB or 50 ll HP0826 or 20 ll GalT. The reactions were started by adding 2 mM UDP-galactose and measurements were taken at 557 nm for 2 min with a time interval of 10 s at different temperatures. The activities were calculated according to the method described in the text. The measurements were done in triplicate and the data shown are the average  standard deviation.

Table 3 Optimal Mn2þ concentration Concentration of MnCl2 (mM)

0

0.05

0.1

0.2

0.5

Relative activity (U/ml)

0.89  0.09 0.67  0.05 7.30  0.17

1.80  0.10 1.02  0.04 12.05  0.11

1.91  0.08 1.18  0.09 19.1  0.12

1.88  0.06 1.33  0.05 17.80  0.08

0.81  0.06 1.11  0.11 16.03  0.29

LgtB (UDP-Gal + GlcNAc) HP0826 (UDP-Gal + GlcNAc) GalT (UDP-Gal + Glc)

The activities were measured in 2 mM phosphate, pH 8.0, 0.01 mM phenol red, 10 mM N -acetylglucosamine, and 50 ll LgtB (or 50 ll HP0826 or 20 ll GalT). The concentrations of MnCl2 were 0, 0.05, 0.1, 0.2, and 0.5 mM, respectively, in the five experiments. The reactions were started by adding 2 mM UDP-galactose and measurements were taken at 557 nm for 2 min with a time interval of 10 s. The activities were calculated according to the method described in the text. The measurements were done in triplicate and the data shown are the average  standard deviation.

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unusually wide range from 10 lM to 10 mM, spanning three orders of magnitude. We set out to determine the optimal concentration of Mn2þ with the method developed. Table 3 shows that both LgtB and GalT have an optimal about 0.1 mM. HP0826, however, has an optimal concentration slightly higher at 0.2 mM. Earlier studies using 10 mM Mn2þ in synthesis reactions may not represent the best conditions under which galactosyltransferase should be used.

Conclusion The developed method based on a pH indicator is sensitive and very easy to use. The method is useful in the initial characterization of galactosyltransferase enzymes, including determination of optimal temperature and cofactor concentrations in biocatalytic synthesis applications, investigation of donor and acceptor substrate specificities in comparison with other similar galactosyltransferases, and screening of potential inhibitors and effectors of the enzymes. The method requires only a small volume of enzyme sample, 10–50 ll or 0.02 U of enzymes in a 1-ml assay format. A typical run takes 2–5 min. Crude enzyme extract can be used without the need for purification. The method requires no expensive enzymes or labeled substrates. Overall, this method is a significant improvement over the current methods that employ radiolabeled materials, sophisticated instruments, and time-consuming procedures. This method is thus widely applicable. The method is amenable for adaptation as a highthroughput screening method where a microtiter plate reader is used. Moreover, with slight adjustment of conditions and with a suitable choice of a pH indicator and a suitable buffer, which takes into account the pH optimal of the enzymes, the method could be easily adapted for other glycosyltransferase enzymes. It should be a valuable tool in glycobiology.

Acknowledgments We thank Dr. Warren W. Wakarchuk and Ms. Melisa Shur of the National Research Council of Canada for providing E. coli constructs overexpressing LgtB and HP0826 and helpful discussions.

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