Objectives

Introduction

Sucrose can be hydrolyzed in the presence of an enzyme called invertase or sucrase.

    Sucrose + H2O ---> glucose + fructose

Invertase is mainly used in the food (confectionery) industry where fructose is preferred over sucrose because it is sweeter and does not crystallize as easily. However, the use of invertase is rather limited because another enzyme, glucose isomerase, can be used to convert glucose to fructose more inexpensively. For health and taste reasons, its use in food industry requires that invertase be highly purified.

A wide range of microorganisms produce invertase and can, thus, utilize sucrose as a nutrient. Commercially, invertase is biosynthesized chiefly by yeast strains of Saccharomyces cerevisiae or Saccharomyces carlsbergensis. Even within the same yeast culture, invertase exists in more than one form. For example, the intracellular invertase has a molecular weight of 135,000 Daltons, whereas the extracellular variety has a molecular weight of 270,000 Daltons.

In contrary to most other enzymes, invertase exhibits relatively high activity over a broad range of pH (3.5--5.5), with the optimum near pH=4.5. The enzyme activity reaches a maximum at about 55ºC. The Michaelis-Menten values of various enzymes vary widely, but for most enzymes K_m is between 2 mM and 5 mM. The Michaelis-Menten value for the free enzyme is typically approx. 30 mM.

Enzyme inhibition is an extremely important area of research in the medical field. For example, lead, mercury, other heavy metals, and nerve gases are extremely poisonous to humans because they are inhibitory to enzymes. For example, Pb^{^++} can easily react with the sulfhydryl (-SH) groups in a protein:

     protein-SH + Pb++ + HS-protein  -----> protein-S-Pb-S-protein + 2H+

The disulfide linkages are critical in creating the three-dimensional structure of a protein, which, in turn, will determine whether or not the enzyme is active. The destruction of the disulfide bonds is definitely detrimental to the enzyme, thus, ultimately harmful to living organisms. There are many sources of environmental pollution. For example, antiknock additives in gasoline contain tetraethyl-lead and other alkyl lead compounds that are released in the atmosphere upon combustion. In 1970, 300,000 tons of lead-based gasoline antiknock additives were released into the air in the U.S. alone; the use of lead is now being phased out gradually.

Although the mechanism of inhibition of invertase by heavy metal ions seems to differ from that just stated, there is no question that invertase, too, is strongly inhibited by heavy metal ions. For example, Ag^{^+} ions attach to the histidine side chains of the invertase molecule and render it inactive. In this experiment, the inhibitory effect of copper sulfate is investigated. At a high concentration, copper sulfate generally imparts unfavorable effects to microorganisms primarily due to inhibition of enzymes. As one example of its use in a laboratory fermentor, the exit gas from the culture can be vented by bubbling it through a solution of copper sulfate to suppress the contaminant from entering into the system.

The effect of another enzyme inhibitor, aniline, is also studied in this experiment. Because of its general ability to inhibit enzyme activity, aniline can be categorized as a poison.

It is important to note that selective enzyme inhibition can also be employed to our advantage. Pesticides or herbicides generally derive their potency from their ability to inhibit enzymes critical for the growth or survival of the organisms. Cures to various diseases are also based on the discovery of enzyme inhibitors. Finally, substrate and product may also themselves act as enzyme inhibitors.

In this experiment, the kinetics of invertase is investigated with the method of initial reaction rates. In this method, the reaction rate can be correlated to the conditions existing at the beginning of the reaction easily, since one has perfect control over the initial condition. The enzyme-substrate mixture is allowed to react for a specified amount of time. The rate of reaction can be easily monitored by measuring the amount of reaction products, i.e., an equimolar mixture of glucose and fructose. The amount of reducing sugars produced is determined colorimetrically with the dinitrosalicylic acid (DNS) introduced in the previous experiment. The task is made easier since the DNS reagent does not react with sucrose.

List of Reagents and Instruments

A. Equipment

• Erlenmeyer flasks
• Beakers
• Graduated cylinder
• Pipets, 0.1 ml, 1 ml, 5 ml
• Test tubes
• Temperature bath
• Thermometer
• Balance
• Spectrophotometer

B. Reagents

• Enzyme
  • Invertase from Baker's yeast, 32 units/mg. (See Note 1.)
  • Stock solution: 1 g/l.
  • (Concentration of the invertase solution to be used in class: 0.04 g/l)
• Sucrose, 50 g/l solution, 200 g/l solution
• Maltose, 50 g/l solution
• Lactose, 50 g/l solution
• Cellulose solution
• Starch solution
• Potassium Phosphate Buffers
  • H₃PO₄ (85% phosphoric acid) (FW=98.00)
  • KH₂PO₄ (monobasic phosphate) (FW=136.1)
  • K₂HPO₄*3H₂O (dibasic phosphate) (FW=228.23)
• Acetate Buffers
  • CH₃COOH (glacial acetic acid) (FW=60.05)
  • CH₃COONa (sodium acetate) (FW=82.03)
• DNS Reagents for the analysis of reducing sugars
• Copper Sulfate Solution, 0.1 M
• Aniline Solution, 0.1 M

Procedures

1. Prepare a working enzyme solution (0.04 g/l) by diluting the stock solution with 0.05 M buffer at pH 7. Prepare various sugar solutions with water.
2. Enzyme Activity versus Enzyme Concentration:
   • Prepare 3 ml enzyme solutions of various concentrations ranging from 0 g/l to 0.04 g/l. One example of how this can be easily prepared is shown in Table 1. First, the amount of 0.04 g/l invertase shown in column #2 is pipetted into each marked test tube, and the amount of buffer solution indicated in column #3 is added to the test tube to dilute the original invertase solution to various levels. To maintain the initial substrate concentration at the same level in each test tube, the volumes are proportioned in such a way as to bring the volume of the resulting invertase solution to 3 ml. Note that Test Tube #A is used to check the background absorbance in the absence of enzyme, and Test Tube #I is used to detect the residual reducing sugar in the enzyme preparation. In addition, Test Tube #J is used to verify whether the addition of DNS reagent indeed stops the hydrolysis reaction.
   • Pre-incubate the solution at the desired temperature for 5 minutes. Although the standard activity is measured at 55ºC, if not specifically requested, room temperature will be used for convenience.
   • After the starting time is noted, 3 ml of 50 g/l sucrose solution is added in quick succession to each of the test tubes marked #A--#J at the same fixed interval (e.g., 10 seconds should be more than adequate. Add 3 ml of DNS reagent to the last test tube marked #J immediately after the enzymatic reaction is initiated.
   • The reaction mixture is allowed to incubate for exactly 5 minutes. Because the initial rate is being measured, the length of reaction must be controlled as accurately as possible.
   • At the end of the incubation period, 6 ml of DNS reagent is added to each of the test tubes in the same order and at the same fixed interval as in Step 1c. This is to ensure that each test tube is incubated for exactly the same length of time. The addition of an alkaline DNS reagent should effectively stop the sucrose hydrolysis reaction. This fact should be indicated by the result from Test Tube #J. Shake each test tube well to mix the reagent.
   • The reducing sugar concentration is measured with the dinitrosalicylic (DNS) colorimetric method. Immerse the test tubes in a 95ºC water bath for 10 minutes to develop the characteristic red-brown color. Measure the absorbance at 540 nm after cooling. Because fructose is also a reducing sugar that reacts with the DNS reagent and turns the solution into a red-brown color, the glucose versus absorbance calibration curve used in the previous experiment is not applicable here. Instead, the amount of sucrose hydrolyzed is calculated from a calibration curve which relates the measured absorbance to an equimolar mixture of glucose and fructose.
3. Effect of Substrate Concentration: Prepare a set of sucrose solutions of various concentrations. The mixtures indicated in Table 2 are suggested. Follow the same experimental procedure to measure the rate of hydrolysis as in the previous step.
4. Effect of pH:
   • Prepare 0.1 M pH buffer solutions ranging from pH=1 to pH=12 in increments of one pH unit. Note that the phosphate buffer is only effective for pH=4.5--9 due to the dissociation constant. Use an acetate buffer to achieve a lower pH. Use monobasic phosphate and phosphoric acid to make a buffer solution of even lower pH. Before coming to the lab, review how to make a pH buffer solution in a freshman chemistry textbook and calculate the relative amounts of KH₂PO₄ (monobasic phosphate) and K₂HPO₄*3H₂O (dibasic phosphate) needed to make these phosphate buffer solutions. Do the same for an acetate buffer.
   • Prepare working invertase solutions (0.04 g/l) by diluting the stock enzyme solution in various pH buffers.
   • Pipet 3 ml of enzyme solutions of increasing pH to test tubes. Table 3 is provided to facilitate the preparation of reaction mixtures. As in the previous steps, mark the time and start the reaction by adding 3 ml of 50 g/l sucrose solution sequentially. React for exactly 5 minutes and determine the rate of reaction colorimetrically.
5. Effect of Temperature:
   • Obtain hot water from either a faucet or a hot temperature bath. Adjust the temperatures of the temporary water baths in 500 ml beakers so that they range from 20ºC to 90ºC in increments of 10ºC. Also prepare cold water baths with ice. See Table 4.
   • Pipet 3 ml of 50 g/l sucrose solution into each of the test tubes and pre-incubate for 5 minutes so that the temperature of the substrate solution is allowed to come to equilibrium with that of the water bath.
   • Follow the procedure outlined previously to start the reaction with the addition of invertase solution and measure the rate of reaction.
6. Effect of Heat Treatment:
   • Pipet 3 ml of 0.04 g/l invertase solution into each test tube indicated in Table 5. Heat-treat the enzyme solution by placing all the test tubes, except one (#A), in a hot (95ºC) water bath. The untreated enzyme is used as the control. Take out the first test tube from the water bath after 5 minutes and quickly bring it to room temperature by immersing it in a cool water bath. Remove the second test tube after 10 minutes, the third after 20 minutes, and so on. Alternatively, heat treatment can be accomplished by placing a sufficient volume of enzyme solution in a hot water bath and withdrawing the desired amount of sample at appropriate times.
   • After the last test tube is withdrawn from heat and cooled to room temperature, add 3 ml of 50 g/l sucrose solution to each of the test tubes. React for exactly 5 minutes as indicated previously.
7. Enzyme Specificity: The specificity of enzyme activity is to be investigated by exposing the enzyme to other disaccharides. Among the commonly occuring disaccharides are maltose and lactose. In place of 3 ml of 50 g/l sucrose solution, use 3 ml of 50 g/l maltose solution or 3 ml of lactose solution as the substrate. Determine if invertase can catalyze the hydrolysis of these disaccharides. Also test cellulose and starch (amylose) solutions. See Table 6.
8. Enzyme Inhibition:
   • Prepare 2 ml copper sulfate solutions of various concentrations ranging from 0.2 mM to 10mM in test tubes. Table 7 suggests some proportions of 0.1 M CuSO₄ solution and water used to achieve this range of concentrations. To each of the test tubes add 1 ml of 50 g/l sucrose solution. Another set of test tubes with a higher range of substrate concentrations can be prepared similarly with a 200 g/l sucrose solution.
   • Note the time and start the reaction by adding 3 ml of 0.04 g/l invertase solution sequentially. React for exactly 5 minutes and determine the rate of reaction colorimetrically.
   • Instead of copper sulfate, repeat the same procedure with aniline as the inhibitor. See Table 8.
   • Follow Step 3 to study the effect of substrate concentration, except that 0.3ml of 0.1 M CuSO₄ is added to each of the test tubes to achieve ~5mM in the final reaction solution.

Notes

1. One international unit of activity is defined as the amount of enzyme needed to hydrolyze 1 µmole of sucrose to invert sugar per minute at pH=4.5 and 55ºC. A stock solution of 1 g/l can be prepared first; dilute the stock solution 1:25 with a buffer to obtain a working solution. If the enzyme preparation does not have the same activity, the concentration of the working solution may be adjusted accordingly with different dilution factors to obtain a final solution of similar activity.
2. Do as many experiments as you wish or as time and supplies/materials permit. You can effectively cover all the procedures by teaming up with a few other classmates and exchanging data at the end of the lab period. (Give the proper credit, or blame for that matter, to your lab partners. Also make sure that you know what your lab partners have done.) However, you must prepare your own lab report.

Questions

1. Based on experimental data, what is the activity of your invertase solution?
2. Plot the enzyme activity versus enzyme concentration. If the relationship is not linear, explain why.
3. Plot the reaction rate versus substrate concentration. Does the reaction follow the Michaelis-Menten kinetics? What are the values of µ_max and K_m?
4. Plot the enzyme activity versus pH. From this curve, what is the optimal pH? Explain why enzyme activity depends on the pH of the solution. Similarly plot the enzyme activity versus temperature. Report the optimal temperature.
5. To what extent did the heat treatment affect the enzyme activity? Is invertase heat-resistant? What happens to an enzyme when it is subjected to heat?
6. Did invertase hydrolyze maltose? Lactose? Cellulose? Starch? Comment on the enzyme specificity.
7. Did the presence of copper sulfate affect the enzyme activities? If so, is it a competitive, a noncompetitive, or an uncompetitive inhibitor? (Base your argument on appropriate plots, e.g., Lineweaver-Burk plots.) Answer the same question for aniline?
8. How would you make an acetate pH buffer solution? List the required chemicals and the composition needed to make one liter of acetate buffer as a function of the pH. Repeat for a citrate buffer. (Phosphate, acetate, and citrate buffers are the most commonly encountered ones.)
9. Report the descriptive name for invertase.
10. Comment on ways to improve the experiment.

Data Forms

             Table 1. EFFECT OF AMOUNT OF ENZYME
     ----------------------------------------------------------
           0.04 g/l     pH=7     50 g/l     Final
     Test  Invertase   Buffer   Sucrose   Invertase  Absorbance
     Tube  Solution   Solution  Solution    Conc.
      #     (ml)       (ml)      (ml)      (mg/l)     (A.U.)
     ----------------------------------------------------------
       A     0.0        3.0       3.0        0.00
       B     0.1        2.9       3.0        0.67
       C     0.5        2.5       3.0        3.33
       D     1.0        2.0       3.0        6.67
       E     1.5        1.5       3.0       10.0
       F     2.0        1.0       3.0       13.3
       G     2.5        0.5       3.0       16.7
       H     3.0        0.0       3.0       20.0
       I     3.0        3.0       0.0       20.0
       J     3.0        0.0       3.0       20.0
     ----------------------------------------------------------

         Table 2. EFFECT OF SUBSTRATE CONCENTRATION
     ----------------------------------------------------------
            50 g/l             0.04 g/l    Final
     Test   Sucrose    Water   Invertase  Sucrose    Absorbance
     Tube  Solution            Solution    Conc.
      #      (ml)      (ml)      (ml)      (g/l)      (A.U.)
     ----------------------------------------------------------
       A     0.0        3.0       3.0         0.00
       B     0.5        2.5       3.0         4.17
       C     1.0        2.0       3.0         8.33
       D     1.5        1.5       3.0        12.50
       E     2.0        1.0       3.0        16.67
       F     2.5        0.5       3.0        20.83
       G     3.0        0.0       3.0        25.00
     ----------------------------------------------------------
     ----------------------------------------------------------
           200 g/l              0.04 g/l    Final
     Test  Sucrose     Water    Invertase  Sucrose    Absorbance
     Tube  Solution             Solution    Conc.
      #      (ml)      (ml)       (ml)      (g/l)      (A.U.)
     ----------------------------------------------------------
       H     0.3        2.7       3.0        10.0
       I     0.5        2.5       3.0        16.7
       J     1.0        2.0       3.0        33.3
       K     1.5        1.5       3.0        50.0
       L     2.0        1.0       3.0        66.7
       M     2.5        0.5       3.0        83.3
       N     3.0        0.0       3.0       100.0
     ----------------------------------------------------------

           Table 3. PH OPTIMUM
     -----------------------------------------
           0.04 g/l      50 g/l
     Test  Invertase     Sucrose    Absorbance
     Tube  Solution      Solution
      #      (ml)         (ml)       (A.U.)
     -----------------------------------------
       A    3.0 (pH= 1)   3.0
       B    3.0 (pH= 2)   3.0
       C    3.0 (pH= 3)   3.0
       D    3.0 (pH= 4)   3.0
       E    3.0 (pH= 5)   3.0
       F    3.0 (pH= 6)   3.0
       G    3.0 (pH= 7)   3.0
       H    3.0 (pH= 8)   3.0
       I    3.0 (pH= 9)   3.0
       J    3.0 (pH=10)   3.0
       K    3.0 (pH=11)   3.0
       L    3.0 (pH=12)   3.0
     -----------------------------------------

          Table 4. TEMPERATURE OPTIMUM
     ----------------------------------------------
                    50 g/l    0.04 g/l
     Test   Temp.   Sucrose   Invertase  Absorbance
     Tube          Solution   Solution
      #     (0C)      (ml)      (ml)       (A.U.)
     ----------------------------------------------
       A      0       3.0       3.0
       B     10       3.0       3.0
       C     20       3.0       3.0
       D     30       3.0       3.0
       E     40       3.0       3.0
       F     50       3.0       3.0
       G     60       3.0       3.0
       H     70       3.0       3.0
       I     80       3.0       3.0
       J     90       3.0       3.0
     ----------------------------------------------

        Table 5. EFFECT OF HEAT TREATMENT
     -----------------------------------------------
          Length of   0.04 g/l   50 g/l
     Test    Heat     Invertase  Sucrose   Absorbance
     Tube Treatment   Solution   Solution
      #     (min)       (ml)       (ml)      (A.U.)
     -----------------------------------------------
       A      0         3.0        3.0
       B      5         3.0        3.0
       C     10         3.0        3.0
       D     20         3.0        3.0
       E     30         3.0        3.0
       F     40         3.0        3.0
       G     50         3.0        3.0
       H     60         3.0        3.0
     -----------------------------------------------

             Table 6. ENZYME SPECIFICITY
     ----------------------------------------------------
                          50 g/l    0.04 g/l
     Test   Substrate    Substrate  Invertase  Absorbance
     Tube                Solution   Solution
      #                    (ml)       (ml)       (A.U.)
     ----------------------------------------------------
       A     Sucrose       3.0         3.0
       B     Maltose       3.0         3.0
       C     Lactose       3.0         3.0
       D     Cellulose     3.0         3.0
       E     Starch        3.0         3.0
     ----------------------------------------------------

                  Table 7. ENZYME INHIBITION (CuSO4)
     ---------------------------------------------------------------------
            0.1 M                 50 g/l   0.04 g/l    Final
     Test   CuSO4       Water    Sucrose   Invertase   CuSO4    Absorbance
     Tube  Solution              Solution  Solution    Conc.
      #      (ml)       (ml)       (ml)      (ml)       (mM)      (A.U.)
     ---------------------------------------------------------------------
       A      0.5        1.5       1.0       3.0        8.33
       B      0.3        1.7       1.0       3.0        5.00
       C      0.2        1.8       1.0       3.0        3.33
       D      0.1        1.9       1.0       3.0        1.67
       E      0.05       1.95      1.0       3.0        0.83
       F      0.02       1.98      1.0       3.0        0.33
       G      0.01       1.99      1.0       3.0        0.17
       H      0.0        2.0       1.0       3.0        0.00
     ---------------------------------------------------------------------
     ---------------------------------------------------------------------
             0.1M                 200 g/l  0.04 g/l    Final
     Test   CuSO4       Water    Sucrose   Invertase   CuSO4    Absorbance
     Tube  Solution              Solution  Solution    Conc.
      #      (ml)       (ml)       (ml)      (ml)       (mM)      (A.U.)
     ---------------------------------------------------------------------
       A      0.5        1.5       1.0       3.0        8.33
       B      0.3        1.7       1.0       3.0        5.00
       C      0.2        1.8       1.0       3.0        3.33
       D      0.1        1.9       1.0       3.0        1.67
       E      0.05       1.95      1.0       3.0        0.83
       F      0.02       1.98      1.0       3.0        0.33
       G      0.01       1.99      1.0       3.0        0.17
       H      0.0        2.0       1.0       3.0        0.00
     ---------------------------------------------------------------------

                Table 8. ENZYME INHIBITION (Aniline)
     ----------------------------------------------------------------------
             0.1 M                50 g/l    0.04 g/l    Final
     Test   Aniline     Water     Sucrose   Invertase  Aniline   Absorbance
     Tube   Solution             Solution   Solution    Conc.
      #       (ml)       (ml)      (ml)       (ml)      (mM)       (A.U.)
     ----------------------------------------------------------------------
       A      0.5        1.5?      1.0        3.0        8.33
       B      0.3        1.7?      1.0        3.0        5.00
       C      0.2        1.8?      1.0        3.0        3.33
       D      0.1        1.9?      1.0        3.0        1.67
       E      0.05       1.95      1.0        3.0        0.83
       F      0.02       1.98      1.0        3.0        0.33
       G      0.01       1.99      1.0        3.0        0.17
       H      0.0?       2.0?      1.0        3.0        0.00
     ----------------------------------------------------------------------
     ----------------------------------------------------------------------
             0.1M                 200 g/l   0.04 g/l    Final
     Test   Aniline     Water     Sucrose   Invertase  Aniline   Absorbance
     Tube   Solution             Solution   Solution    Conc.
      #       (ml)       (ml)      (ml)       (ml)      (mM)       (A.U.)
     ----------------------------------------------------------------------
       A      0.5        1.5?      1.0        3.0        8.33
       B      0.3        1.7?      1.0        3.0        5.00
       C      0.2        1.8?      1.0        3.0        3.33
       D      0.1        1.9?      1.0        3.0        1.67
       E      0.05       1.95      1.0        3.0        0.83
       F      0.02       1.98      1.0        3.0        0.33
       G      0.01       1.99      1.0        3.0        0.17
       H      0.0?       2.0?      1.0        3.0        0.00
     ----------------------------------------------------------------------

                Table 9. ENZYME INHIBITION (CuSO4)
     -----------------------------------------------------------------
             50 g/l    0.1 M            0.04 g/l    Final
     Test   Sucrose    CuSO4     Water  Invertase  Sucrose  Absorbance
     Tube   Solution  Solution          Solution    Conc.
      #       (ml)      (ml)      (ml)    (ml)      (g/l)      (A.U.)
     -----------------------------------------------------------------
       A      0.0       0.3       2.7     3.0        0.00
       B      0.5       0.3       2.2     3.0        4.17
       C      1.0       0.3       1.7     3.0        8.33
       D      1.5       0.3       1.2     3.0       12.50
       E      2.0       0.3       0.7     3.0       16.67
       F      2.5       0.3       0.2     3.0       20.83
       G      2.7       0.3       0.0     3.0       22.50
     -----------------------------------------------------------------