Measurement of Enzyme Kinetics by Use of a Blood Glucometer

Laboratory Experiment pubs.acs.org/jchemeduc

Measurement of Enzyme Kinetics by Use of a Blood Glucometer: Hydrolysis of Sucrose and Lactose Peter Heinzerling,* Frank Schrader, and Sascha Schanze Institute for Science Education−Chemistry Education, Leibniz Universität Hannover, Im Kleinen Felde 30, D-30167 Hannover, Germany S Supporting Information *

ABSTRACT: An alternative analytical method for measuring the kinetic parameters of the enzymes invertase and lactase is described. Invertase hydrolyzes sucrose to glucose and fructose and lactase hydrolyzes lactose to glucose and galactose. In most enzyme kinetics studies, photometric methods or test strips are used to quantify the derivates of the substrates. The use of a commercial blood glucose meter to determine the hydrolyzed glucose is described. This inexpensive and efficient method can be used when teaching enzymatic kinetics in lower-level biochemistry laboratories.

KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Bioanalytical Chemistry, Carbohydrates, Enzymes, Food Science, Kinetics, Laboratory Equipment/Apparatus

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n alternative analytical method is described for measuring the kinetic parameters of the enzymes invertase and lactase. Invertase hydrolyzes sucrose to glucose and fructose and lactase hydrolyzes lactose to glucose and galactose. In most enzyme kinetics studies, photometric methods or test strips are used to quantify the derivates of the substrates. The use of a commercial blood glucose meter to determine the hydrolyzed glucose is an inexpensive and efficient method and can be used when teaching enzymatic kinetics in lower-level biochemistry laboratories. In this Journal, Perles and Volpe suggested measuring the concentration of glucose during its mutarotation with a simple and inexpensive blood glucometer.1 In a following article, Hardee, Delgado, and Jones presented an accurate and broad assay to obtain the kinetic parameters for the noncatalyzed and enzyme-catalyzed reaction of mutarotation.2 The blood glucometer allows the measurement of concentrations of D-glucose in solvent within 5 s. The test sensors are sophisticated high-tech products with integrated nanoscaled membranes and detectors. The accuracy of the results with whole blood was checked and evaluated during the process of accreditation.3,4 In aqueous systems, the results cannot be measured correctly on a quantitative basis because the sensors are calibrated in a blood matrix. However, the results are consistent and for the determination of exact values, a correction factor can be calculated using glucose standard solutions.2 A basic laboratory experiment was developed for a general biochemistry course. The experiments involved a common and simple measuring system and produced direct results. © 2012 American Chemical Society and Division of Chemical Education, Inc.

GLUCOMETER

The sensors used by Perles and Volpe contained the enzyme− coenzyme system GDH/PQQ5 combined with the Roche Diagnostics AccuChek meter. The sensors of Hardee, Delgado, and Jones used GOD/NAD6 combined with a meter from LifeScan, a Johnson & Johnson company. The enzymes oxidize β-glucose to gluconic acid via gluconolactone. The meter shows the total glucose concentration in mg dL−1 or mmol dL−1 using amperometry where the electron flow current during the redox reaction is proportional to the concentration of glucose. Potassium ferricyanide is used as the redox mediator7 to obtain the current flow (Figure 1). GDH/PQQ showed interferences with galactose, maltose, and xylose. For this reason, Roche Diagnostics modified their technology in 2011, now using a mutated enzyme. The new sensors were tested; however, the accuracy of the results in aqueous systems was unacceptable.

Figure 1. Reaction mechanism of GOD-based blood glucometer. Published: October 2, 2012 1582

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alternative experiment is enzymatic hydrolysis with invertase from baker’s yeast. Miloski et al. compared both hydrolyses, using strong acid and invertase, by investigating ingredients of drinks.8 Originally, this reaction was the source of the theory of Michaelis and Menten in 1913;9 they called the enzyme invertin. The systematic name is β-fructofuranosidase (EC 3.2.1.26) and it is produced for the sweets industries from Saccharomyces cerevisiae. The enzyme cleaves the glycosidic linkage of the sucrose molecule. The theory of Michaelis and Menten is only valid with a large excess of substrate relative to the enzyme. The theory refers to the reaction between enzyme (E) and substrate (S) to give the enzyme−substrate complex (ES), which dissociates into enzyme (E) and product (P):

For the experiments described here, the OneTouch Vita system from LifeScan with GOD/NAD was used, a system that is available worldwide and is identical to that used by Hardee, Delgado, and Jones.2 Within the sensor, there is a double electrode system to control the accuracy of the results. The meters must be calibrated with standard aqueous solutions before starting experimental procedures. When inserted into the solution, the sensors automatically absorb a sample of 0.6 μL within a second and the meter produces the result within 5 s. Up to 500 results can be stored in the meter and could be transferred to a computer. The calibration of the sensors is shown in Figure 2. Each measurement per experiment was

k+1

k+2

k −1

k −2

E + S XooY ES XooY E + P

(1)

Under steady-state conditions and saturation of the enzyme with substrate, the equation with initial rate v0 leads to v0 =

Vmax ·[S] KM + [S]

(2)

where Vmax is the maximal initial rate v0 that enzyme can achieve and K M is the Michaelis constant with the concentration of the substrate at 1/2Vmax. Lineweaver and Burk10 suggested the double-reciprocal form:

Figure 2. Calibration of glucose in an aqueous solution with ONETOUCH Vita blood glucose monitoring system.

K + [S] K 1 1 1 = M = M· + v0 Vmax ·[S] Vmax [S] Vmax

repeated five times under the same conditions. Within the experiments, the standard deviations were between 0.9 and 2.2%. The concentration of glucose should be in a range of 20− 600 mg dL−1. If the results are beyond the upper range, the assay has to be diluted. This was carried out in all reported experiments with measured glucose concentrations higher than 500 mg dL−1.

(3)

The plot of 1/v0 against 1/[S] is the Lineweaver−Burk plot. For 1/v0 = 0, the intercept gives −1/KM and the 1/v0 intercept gives the value of 1/Vmax . There are two methods for determining the concentration of the product [P] to obtain v0: continuous and discontinuous. A typical discontinuous method with the end-point assay, where [P] is measured after a fixed time. This can be done under linearity conditions of [P] versus time. This was controlled for both reactions: the result of the [P] versus time plot were linear functions during first 60 min (Figures 3 and 7). In a first step, the results of [P] were adapted to the calibration of the probes, and in a second step, they were adapted to the function of the linearity test. The adapted results are indexed with an asterisk. The reciprocal form of Lineweaver−Burk can be transformed with the adapted initial rate v0*:

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EXPERIMENTAL OVERVIEW The experiments were performed in a bioanalytical lab with grade-13 students and with upper-level undergraduate students. The students work in pairs to conduct a series of experiments to determine (i) the Michaelis constant KM as a result of v0 versus [S], (ii) the optimum pH as a result of v0 versus pH, and (iii) the optimum temperature as a result of v0 versus temperature. Two series of experiments are presented: the enzymatic hydrolysis of sucrose with invertase and the enzymatic hydrolysis of lactose with lactase. Students obtain experimental data by assessing the glucose concentration in the reaction mixture using the glucometer 30 min for the hydrolysis of sucrose and 20 min for the hydrolysis of lactose after the enzymatic hydrolysis was initiated. The two series of experiments can each be completed in a three-hour laboratory.

v0* =

[P]* t

(4)

K + [S] KM 1 1 1 = M = · + v0* Vmax*·[S] Vmax* [S] Vmax*

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(5)

HAZARDS No hazardous materials were used in any of the experiments.

As a result of this adaption, −1/KM at 1/v0* = 0 is of the same value. The slope will change to KM/Vmax* and the 1/v0* intercept gives 1/Vmax*.

THEORETICAL BACKGROUND The hydrolysis of sucrose in strong acid is a common experiment in the undergraduate laboratory. Its kinetics is determined by polarimetry based on the different optical rotations of sucrose and the products glucose and fructose. The reaction is frequently known as inversion of sucrose. An

ENZYMATIC HYDROLYSIS OF SUCROSE To introduce the students to Michaelis−Menten kinetics, the hydrolysis of sucrose with invertase contained in dried yeast was investigated. A suspension of dried baker’s yeast (0.5 g per 100 mL H2O) and a stock solution of sucrose (20 g per 100 mL H2O) were prepared. The stock solution was diluted to make

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five different solutions (10.00, 6.67, 5.00, 3.33, 2.50 g dL−1) to investigate the kinetic parameters of Michaelis and Menten. All measurements were performed at 22 °C and the sucrose was incubated with a phosphate buffer (see below) of pH = 7.0 for 30 min. The hydrolysis was initiated by the addition of 1 mL of the yeast suspension to 9 mL of each substrate dilution (5 mL sucrose plus 4 mL buffer). Medical syringes and Eppendorf micropipets were used to ensure volume measurement with an adequate precision. The concentrations of sucrose, taking the added volumes of buffer and enzyme into account, are listed in the Supporting Information.

be explained by environmental factors such as substrate, salt concentration, purity, and the presence of other enzymes in yeast as there are fermentation enzymes within yeast that can react with sucrose and glucose. Also, the diffusion and transport of sucrose into yeast as well as glucose consumption and its diffusion and transport outside of yeast influence kinetic parameters. Low-cost unpurified dried yeast-extract was used and tested with a blank test and no glucose was displayed. Published data for unpurified invertase from yeast was not found. Relative to the goal of finding a simple approach to obtain the Lineweaver−Burk plot in a basic biochemistry course, the experiments were successful. The experiments are of low complexity and results can be achieved within a 90-min laboratory period. The cost is $6.50 per group of students.

Determination of Michaelis Constant

To obtain the data for a Lineweaver−Burk plot, the initial reaction rate v0 was calculated from the measured concentrations of every sample after 30 min. This end-point assay is commonly used in photometric measurements. The assay needs only one sensor per result, whereas the continuous method used by Hardee et al.2 needs three to four. The linearity of the results was tested separately (Figure 3) and

pH Optimum

Phosphate buffers (Sørensen buffers: NaH2PO4 0.066 M, K2HPO4 0.066 M) were prepared to achieve the pH levels 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 and standard acetate buffer was prepared to achieve pH 4.0. A mixture of 4 mL of buffer and 1 mL of yeast suspension was prepared for each pH level and incubated at room temperature for 20 min. An addition of 5 mL of sucrose stock solution initiated the reactions. The glucose concentration was measured after 15 min and converted into rate units (Figure 5). The enzyme shows two

Figure 3. Linearity test of the hydrolysis of sucrose with invertase from dried yeast.

corrected them using the calibration factor. Both plots, the original and corrected, were placed within the graph to show that the correction only influences the 1/v0 term (Figure 4) and is in line with the theory of Michaelis−Menten. The standard deviations were within a range of 1.2−3.6%. KM was found to be 331 ± 10 mmol/L. Results from purified invertase are reported in the literature as KM = 150 mmol/ L.11,12 The difference between literature and these results could

Figure 5. Plot of rate versus pH (T = 295 K) showing the enzymatic hydrolysis of sucrose.

maxima, one at pH 4.5 and the second at a range between pH 6.0 and 7.5. The standard deviations were between 1.4 and 3.4%. This is in accordance with the literature.19 The highest activity is stated in literature at pH 4.5. These results can be reached within a 45-min laboratory period. Temperature Optimum

This experiment was conducted at pH = 7.0. All solutions were heated in electronically controlled water baths. Five milliliters of the stock solution of sucrose and a mixture of 4 mL of buffer with 1 mL of yeast suspension were incubated for 15 min. Fifteen minutes after mixing the two solutions, the glucose concentration was measured and converted into rate units (Figure 6). The probes between 40 and 55 °C must be diluted 1:4 before measurement. The temperature optimum of 45 °C is in accordance with the literature.11,19 The standard deviations were between 1.7 and 14%, with a majority of less than 5%. Having good time management, results can be achieved within a 45-min laboratory period.

Figure 4. Lineweaver−Burk plot of the hydrolysis of sucrose (pH = 7.0, T = 295 K): original data (blue circles) and adapted data (pink diamonds). 1584

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Figure 6. Plot of rate against temperature (pH 7.0) showing the enzymatic hydrolysis of sucrose.

Figure 7. Linearity test of the hydrolysis of lactose with lactase from capsules.

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ENZYMATIC HYDROLYSIS OF LACTOSE In this Journal, several articles have described the enzymatic hydrolysis of lactose by enzyme lactase.13−18 Lactose is hydrolyzed into α-glucose and β-galactose; α-glucose and βgalactose equilibrate with β-glucose and α-galactose, the socalled mutarotation. The enzyme lactase (β-galactosidase, EC 3.2.1.26) cleaves the glycosidic linkage of β-galactose in the lactose molecule. The use of commercial lactase in solutions has been reported;13−15 capsules or tablets are available from drug stores. Three of the publications described the analysis of the enzyme activity via determination of the reaction product glucose; in two of them, GOD enzyme assays13,17 were used, whereas the other conducted measurements with test strips.13 A blood glucose meter was used as described above. This method is as fast as the use of test strips, but more accurate according to the obtained data. The initial rate of lactase can be calculated after 20 min.

Figure 8. Lineweaver−Burk plot of the hydrolysis of lactose (T = 295 K, pH 7.0): original data (blue diamonds) and adapted data (pink diamonds).

Determination of Michaelis Constant

In the experiments, a suspension of lactase from Aspergillus oryzae was prepared using capsules (5 capsules per 5 mL of H2O). The capsules have defined ingredients without the other carbohydrates. The suspension should be prepared at least one day before experimental procedures and stored in a refrigerator. Lactose monohydrate, 5 g/100 mL H2O, was used as a stock solution, which is comparable to the concentration in whole milk. A dilution series of lactose (3.333, 2.500, 1.667, 1.000, 0.667 g dL−1) was prepared to get the Lineweaver−Burk plot. All experiments were conducted at room temperature and pH = 7.0. The hydrolysis of the solutions was initiated by the addition of 0.1 mL of the lactase suspension. The lactase suspensions were portioned with an Eppendorf micropipet. A linearity test shows a typical presteady-state run with best conditions for an end-point assay between 20 and 30 min (Figure 7). To obtain the data for the Lineweaver−Burk plot, the initial reaction rate of all samples was calculated after 20 min (Figure 8). The slope of the line was obtained by calculating the linear regression function. The standard deviations of the regression function were in a range of 0.6 to 2.4%. The result for KM = 70 ± 2 mmol/L is slightly lower than the published value,20 where KM = 85 mmol/L at unknown pH was reported. Single KM matched the literature value. The experiment was repeated with unbuffered regular milk (Figure 9). The standard deviations of the regression function were in a range of 0.8−3.2%. The results were KM = 73 ± 2

Figure 9. Hydrolysis of lactose using regular milk (T = 294 K, pH = 6.8): original data (blue circles) and adapted data (pink diamonds).

mmol/L. This is slightly lower than the published constant of 85 mmol/L.20 pH Optimum

Acetate and phosphate buffers were prepared to achieve the pH levels of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. A mixture of 9 mL of buffer and 1 mL of the stock solution was incubated at room temperature for 20 min. Reactions were started with 0.1 mL enzyme suspension at intervals of 2 min. The glucose concentration was measured after 20 min (Figure 10). The pH optimum of about pH = 4.5 is in accordance with the literature.21,22 The standard deviations were in a range of 1.0− 3.9%. 1585

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ACKNOWLEDGMENTS Sabine Klevenz, Marketing Manager at Lifescan Germany, a Johnson & Johnson Company, supported us with some materials. We thank Fonds der Chemischen Industrie (FCI) Germany for financial support. We also thank Detlef W. Bahnemann (Institute of Technical Chemistry) for the use of his laboratory and his helpful and inspiring discussions.

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Figure 10. Rate versus pH of the hydrolysis of lactose (T = 294 K).

Temperature Optimum

In these experiments, phosphate buffers of pH 7.0 were used. All solutions were heated with electronically controlled water baths. For each replicate, 10 mL of buffer solution was mixed with 0.1 mL of lactase suspension and incubated for 15 min separately. Reactions were started with 1 mL of lactose stock solution with intervals of 2 min. The glucose concentration was measured after 10 min (Figure 11). The temperature optimum of 45 °C is in accordance with the literature (T = 45 °C).21,22 The standard deviations were in a range of 1.9−10.9%, most of them near 5%.

Figure 11. Rate versus temperature plot of the hydrolysis of lactose (pH 7.0).

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CONCLUSIONS The students were impressed by the ease of the method making use of the blood glucometers. Future studies will expand the experiments on other over-the-counter enzyme products from pharmacies.

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REFERENCES

(1) Perles, C. E.; Volpe, P. L. O. J. Chem. Educ. 2008, 85 (5), 686− 688. (2) Hardee, J. R.; Delgado, B.; Jones, W. J. Chem. Educ. 2011, 88 (6), 798−800. (3) Evaluation Report: AccuChek Aviva System, Roche Diagnostics 2007; https://www.poc.roche.com/en_US/pdf/Aviva_Evaluations_ White_Paper.pdf (accessed Aug 2012). (4) Young, J. K.; Ellison, J. M.; Marshall, R. Performance Evaluation of a New Blood Glucose Monitor That Requires No Coding: The OneTouch Vita System, J. Diabetes Sci. Technol. 2008, 2(5), 814-818. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2769794/ (accessed Aug 2012) (5) GDH/PQQ is glucose dehydrogenase-pyrrolquinoline quinone. Since 2003, the accepted name has been Quinoprotein Glucose Dehydrogenase (EC 1.1.5.2). (6) GOD/NAD is glucoseoxidase-nicotinamide adenine dinucleotide. (7) Kitamura, T.; Kaimori, S.; Harade, A.; Ishikawa, T.; Fujimura, T.; Nakamura, H.; Gotoh, M.; Karube, I. SEI Tech. Rev. 2006, 63, 19−22. (8) Miloski, K.; Wallace, K.; Fenger, A.; Schneider, E.; Bendinskas, K. Comparison of Biochemical and Chemical Digestion and Detection Methods for Carbohydrates. Am. J. Undergrad. Res. 2008, 7 (2), 7−18. (9) Michaelis, L.; Menten, M. Biochem. Z. 1913, 49, 333−369. (10) Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 65, 658. (11) Akgöl, S.; Kaçar, Y.; Denizili, A.; Arica, M. Y. Food Chem. 2001, 74 (3), 281−288. (12) Tanriseven, A.; Dogan, S. Process Biochem. 2001, 36 (11), 1081−1083. (13) Melton, T. J. J. Chem. Educ. 2001, 78 (9), 1243. (14) Russo, S. F.; Moothardt, L. J. Chem. Educ. 1986, 63 (3), 242− 243. (15) Pope, S. R.; Telleson, T. D.; Williams, R. J.; Underhill, R. D.; Deal, S. T. J. Chem. Educ. 1998, 75 (6), 761. (16) Allison, M. J.; Bering, C. L. J. Chem. Educ. 1998, 75 (10), 1278− 1280. (17) Mullis, T. C.; Winge, J. T.; Deal, S. T. J. Chem. Educ. 1999, 76 (12), 1711. (18) Harris, D.; Seefeldt, L. J. Chem. Educ. 2009, 86 (11), 1271. (19) Beta-fructofuranosidase on Brenda. http://www.brendaenzymes.org/php/result_flat.php4?ecno=3.2.1.26 (accessed Aug 2012). (20) Beta-galactosidase on Brenda. http://www.brenda-enzymes.org/ php/result_flat.php4?ecno=3.2.1.23 (accessed Aug 2012). Crueger, A; Crueger, W. Biotechnology; Kieslich, K., Ed.; Verlag Chemie: Weinheim, Germany, 1984; pp 421−457. (21) Tanaka, Y.; Kagamiishi, A.; Kiuchi, A.; Horiuchi, T. J. Biochem. 1975, 77 (1), 241−247. (22) Kilara, A.; Shahani, K. M.; Wagner, F. J. Food Biochem. 1977, 261−273.

ASSOCIATED CONTENT

S Supporting Information *

Student handout and instructor information. This material is available via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 1586

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