EXPERIMENT 3 ENZYMATIC QUANTITATION OF GLUCOSE This is a team experiment. Each team will prepare one set of reagents; each person will do an individual unknown and each team will submit a single report. A. DATA STORAGE Bring a USB storage device to collect data. B. OVERVIEW Glucose oxidase (GO). Enzymes are protein catalysts with very fine mechanism of action which ensures high selectivity (image: RCSB) There are 4 million people with diabetes in the United States. For many of them, constantly monitoring the blood sugar level is a matter of staying alive. One of the commercial techniques for measuring the blood glucose level is based on the use of the enzymatic reaction involving glucose oxidase. Here we demonstrate this analytical technique in the laboratory setting. The highlights of the experiment are: The spectrophotometer is used for the first time to quantitatively determine the concentration of the analyte. Using the absorbance readouts from the spectrophotometer, we monitor the course of the chemical reaction in real time. By running measurements on a series of calibration samples, we construct a calibration plot that is subsequently used to determine the concentration of unknown. By probing the response to different analytes, we characterize the selectivity of the method. The conformation of glucose before cleavage by glucose oxidase C. REACTIONS Glucose oxidase (GO) catalyzes the oxidation of glucose, followed by hydrolysis which leads to production of the D-gluconic acid: GO - -glucose -gluconic acid β D + O + HO D + H O (Slow) (1a) Hydrogen peroxide produced by reaction 1a can be detected in a variety of ways. In this experiment the hydrogen peroxide is detected using the molybdate-catalyzed reaction with iodide as follows Mo( VI ) + H O + 3 I + H I 3 + H O (Fast) The triiodide produced by this latter reaction is detected spectrophotometrically by its absorption at 360 nm. A sufficient amount of catalyst, Mo(VI), is used that reaction 1b is much faster than reaction 1a. Accordingly, the rate of production of triiodide reflects the rate of reaction 1a. (1b) 1
D. MEASUREMENT APPROACH You will use a computer to collect data for absorbance vs. time. For reasons to be discussed later in this course, the change in absorbance, A, during a fixed time interval, t, is expected to vary linearly with glucose concentration. You will prepare and use a calibration plot of absorbance, A 100, vs. glucose concentration, C G, where A 100 is the absorbance after a 100-s reaction time and C G is the glucose concentration. Timing during the measurement step is critical to the success of this experiment. E. EXPERIMENTAL You will work as groups to prepare and run standards and to prepare a report. Each person should process an unknown and report the results. Use distilled water to prepare all solutions. 1. Solutions provided a. Stock glucose solution: A solution containing approximately 1.00 g of glucose (MM = 180. g/mol) per liter will be provided. Record the exact concentration (g/l) of glucose in the solution. b. Stock galactose solution: A solution containing approximately 0.1 g of galactose (MM = 180. g/mol) per 50 ml will be provided. Record the concentration of galactose in the solution. c. Buffer-catalyst solution: A solution containing 3.4 g of (NH 4 ) 3 Mo 7 O 4.4H O per liter of phosphate buffer solution will be provided. The phosphate buffer solution is prepared by dissolving approximately 0.5 g of KH PO 4 (MM = 136) and 10.7 g of K HPO 4 (MM = 8.) per liter of solution and adjusting the ph to 6.5.. Solutions you must prepare All dilutions are to be made using distilled water. One 100-mL volume of solution (f) should be sufficient for all groups in each laboratory. Each group should prepare only one set of solutions a, b, c, d, g, h. Each person is responsible for his/her unknown. a. Diluted glucose stock solution: Add 5.00 ml (transfer pipet) of the stock glucose solution to a 50-mL volumetric flask dilute to volume, and mix thoroughly by repeated inversions. b. Diluted glucose standards: Use a buret to add 3.50, 7.00, 10, 14.0 and 17.5 ml of the diluted stock solution to each of five 100-mL volumetric flasks, dilute each to volume and mix thoroughly. Calculate and report the molar concentrations of these solutions. c. Diluted galactose standard: Pipet 10.00 ml of the galactose solution into a 100-mL volumetric flask, dilute to volume and mix thoroughly. Calculate the molar concentration. d. Mixture of galactose and glucose: Add 10.0 ml of the diluted galactose solution and 10.0 ml of the diluted stock glucose solution to a 100-mL volumetric flask, dilute to volume and mix thoroughly. e. Unknown solution: Your unknown will be provided in a 50-mL volumetric flask. Dilute the solution to volume with distilled water and mix thoroughly. f. Glucose oxidase solution: Weigh 0.4 g of glucose oxidase (3 x 10 3 U/g) to the nearest 0.01 g, dissolve in water and dilute to 100 ml. (Handled carefully, one solution should be sufficient for all groups in a room.) g. Potassium iodide solution: Dissolve 40.3 g of potassium iodide in water and dilute to about 1 L. h. Composite reagent: Add 40 ml of the buffer-catalyst solution, 40 ml of the potassium iodide solution and 10 ml of the glucose oxidase solution to a 100-mL volumetric flask, dilute to volume, and mix thoroughly. Permit this reagent to warm to room temperature before using it.
3. Measurement step a. Instrumentation: You will use a Genesys spectrophotometer interfaced to a computer for this experiment. Turn on the spectrophotometer at least 0 min before you plan to use it. The spectrophotometer must be on long enough to see an absorbance reading in the display of the instrument before continuing with the next step. This usually takes -3 minutes. Cuvet being placed into Genesys spectrophotometer b. Computer settings: Click on the Spec 0Gen Abs Kinetics icon on the desktop. Once the program loads, the software will take control of the spectrophotometer. The display of the spectrophotometer will read, Computer Controlled Do not press any keys c. Procedure: Add exactly 5.00 ml (buret) of the composite reagent to each of seven clean, dry spectrophotometer cuvets plus one additional cuvet per group member. Cover each cuvet tightly with Parafilm. From this point on, process each sample independently. You are approaching the most critical part of this experiment; read the notes below before proceeding. NOTES: It is very important that the mixing step described below be completed as rapidly as possible, within 5 seconds, if possible. Learning to use the syringe properly is one of the most critical steps in this experiment. It is suggested that one person from each group practice using the syringe several times before starting to make runs. A good way to practice is to tare a weighing bottle on a balance and weigh several -ml injections of distilled water without removing the bottle. (With practice, you should be able to inject volumes reproducible to within about 0.01 ml.) Rinse the syringe at least four times with each new solution before making injections. i. Blank solution: Use a syringe to add exactly.00 ml of water to one of the cuvets containing 5.00 ml of composite reagent. Mix thoroughly and use this as the blank solution. ii. Software Usage: The software should be loaded by this time. See part b, above. Click on the Set Up button which will open the Calibrate window. Set the wavelength to 360 (if not already set). Put the cuvet with the blank solution into the holder, close the cover and click the zero button. Doing so allows the software to set the spectrophotometer to a reading of 100 %. The word uncalibrate disappears. Click OK to get back to the main program. In the lower right-hand portion of the window set the number sec/point to 4. The instrument is now ready to use. iii. Standards and unknown: Use the -ml syringe to inject.00 ml of the lowestconcentration standard through the Parafilm into the composite reagent in a cuvet. (Direct the needle toward the side so that air bubbles are not carried into the solution.) Immediately start a stop-watch, place your thumb over the top of the cuvet and invert it several times to ensure complete mixing. Place the cuvet into the cuvet well in the spectrophotometer, close the cover and click the 3
start button on the computer screen. Data acquisition should be initiated at exactly the same time after injection of each sample (for example, 8 seconds after the injection). Collect approximately 40 data points (40 x 4 s/point = 160 s) for each sample and click Stop iv. Saving your data: Click Save, make sure that the Absorbance box and boxes for any other values you want to save are checked and proceed to save the data on your storage device using a suitable file name. v. Exiting the program: Once you are finished using the software, exit the program. Repeat steps ii and iv for each standard, unknown, the diluted galactose sample, and the mixture of galactose and glucose. Don t forget to save your data after each run. F. DATA PRESENTATION/PROCESSING USING ORIGIN Parts of the Origin tutorial that will be useful are the FORMATTED TEMPLATE, (Part II-J) and instructions for using the least-squares tool (Part III). 1. Plots of absorbance vs. time Prepare a single figure containing formatted plots of absorbance vs. time data for each standard and the diluted galactose sample. Use lower-case letters to label the six plots on the figure and include identifying information (concentration) in the figure legend. Include this figure as Figure 1 in your report. Prepare a figure containing plots of absorbance vs. time for a) the solution containing 10.0 ml of glucose and b) the solution containing 10.0 ml each of galactose and glucose solutions. Include this plot as Figure in your report. Compare the slopes of the two plots and discuss the significance of the result. NOTE: Origin is recommended. As the columns should be the same, the easiest way to combine several plots onto one graph using Origin is to put one set of time data in Column A and absorbance data in adjacent columns (e.g. Columns B G). Highlight appropriate columns (e.g. B G) containing absorbance data and click Plot/Scatter ; six plots of absorbance vs. concentration should appear on the same graph. You can save formatting time by using the FORMATTED TEMPLATE prepared in the Origin tutorial (Part II-J).. Absorbance vs. concentration (glucose data only) a. Tabular data: Read the absorbances at t = 1 s and t = 100 s from your data columns for each standard, unknown and the diluted galactose standard. Tabulate these data and calculate the absorbance difference, A = A 100 A 1. Include this table in your report. b. Graphical data: Prepare a calibration plot of absorbance difference, A, vs. glucose concentration (10-5 mol/l) and do a least-squares fit of the data. (Do not include galactose data on the calibration plot or in the least-squares fit!) Include a properly formatted plot including experimental data and the least-squares line as Figure 3 in your report. Hint: a) You can save time by using the FORMATTED TEMPLATE prepared as part of the Origin tutorial (Part II-J). b) See the Origin tutorial (Part III-B) for least-squares procedures, including re-plotting procedures (Part III-B-3). c. Least-squares results: Include the least-squares statistics in your report. In this and all subsequent experiments, least-squares statistics should include the slope and its standard deviation, the intercept and its standard deviation, the standard error of the estimate and the correlation coefficient. 4
A convenient way to present least-squares equations with appropriate statistics is as follows Y = α ± s + ( β ± s ) X with syx = value and r = value (a) α β in which α and β are the intercept and slope, s α and s β are the standard deviations of the slope and intercept, s yx is the standard error of the estimate, and r is the correlation coefficient. The format with some typical numbers in it is 3 A = ( 1.35 ± 3.) 10 + (8.6 ± 0.19) 10 C with s 0.0015 and glucose yx = r = 0.986 (b) The units are: au (absorbance units) for A and s α and au for s yx. You may want to use this format for future reports. α ±, M (mol/l) for C, au/m for glucose β ± s β, 3. Unknown concentration Use the least-squares parameters and absorbance difference, A, of your unknown to calculate the molar glucose concentration in the 50-mL volumetric flask after dilution to volume. Also calculate the standard deviation and 95 % confidence interval for the unknown concentration. (Hint: Procedures for standard deviation and confidence interval are discussed in the text and lecture; see also section H below) You should report your results as instructed the week after the experiment is completed as well as in your written report. 4. Galactose sample By checking an organic text, you will observe that glucose and galactose have very similar structures. Using the results of this experiment comment on the selectivity of glucose oxidase for glucose relative to galactose. Does the presence of galactose in a glucose sample have a significant effect on the rate of change of absorbance with time? G. REPORT In addition to information specified in the course packet, your report should include the following. A figure containing plots of absorbance vs. time for the standards and the galactose sample. A figure containing plots of absorbance vs. time for the 10.0-mL glucose sample and the sample containing a mixture of galactose and glucose. Tabular data for A 0, A 100, A your unknown and the galactose sample. A figure containing the calibration plot including least-squares line. Equations or tabular data for least-squares statistics (A vs. C (10-5 M)). A discussion of the selectivity of glucose oxidase for glucose in the context of similarities between structures for glucose and galactose. 5
H. REFERENCE INFORMATION ON DATA ANALYSIS Since the report needs to be written before certain statistics topics are discussed in class, some reference information is supplied in this section. The slope and the intercept of the linear regression together with their statistical errors, β ± s β and α ± s α, are evaluated and reported by Origin. The formulas used toward this end are Eqs. 8-13, 8-14, 8-16, and 8-17 in the text. Note that in the text the slope is denoted m ( = β ) and the intercept is denoted b ( = α). Note that Eq. 8-16 should read s = s S. m r xx The standard error of the estimate, s yx, is alternatively referred to as standard deviation about the regression. In the text it is denoted as s r and given by Eq. 8-15; Origin uses the abbreviation SD for this parameter. s r is closely related to the residual, s = SS ( N ), see Eq. 8-19. The correlation coefficient r ( = R) is defined by Eq. 8-1. Finally, how should one report the error in the concentration of unknown? To determine the error, one should first evaluate the parameter s c as given by Eq. 8-18. In this expression M is the number of replicate measurements of the unknown. In our experiment M = 1 since a single A value is acquired for the unknown. The error should be reported as ± tsc, where the coefficient t is determined from the Tab. 7-3 in the text for the requested confidence interval of 95% assuming that the number of degrees of freedom is r resid N (two degrees of freedom are consumed to determine the slope β and the intercept α ). In-depth discussion of t will be presented in class. Updated 1/5/09 6