Laboratory Class 3: Enzymes 2026
BCH 2333 Lab 3: Enzymes Study Guide with Complete Solution | 2026 Update
PROCEDURES
Several aspects of the kinetics discussed above will be studied in this lab session, using a
commercial acid phosphatase preparation from wheat germ. First of all, you will investigate the
time dependence of the enzyme catalysed reaction. Following measurement of the initial rates
of reaction with fixed amount of phosphatase and variable concentrations of substrate in the
presence and absence of an unknown inhibitor, Hanes plots will be constructed, and KM and Vmax
will be determined from these plots. The analysis of the effect of the inhibitor on the kinetic
parameters of the enzyme will allow you to identify the type of inhibitor that you have used.
EXPERIMENT #1: Rate of product formation
As pointed out in the discussion of initial velocity the acid phosphatase assay must be carried out
under conditions where the rate of product formation is constant over the incubation period.
This is critical because the assay is a fixed time assay, and the enzyme is subject to product
inhibition. In order to check the rate of product formation, a time curve will be constructed. Each
student will use different amounts of enzyme, thus with the results of the whole group, you will
be able to analyze the dependence of the enzyme activity on the enzyme concentration.
In this experiment, you will be handling a rack with assay tubes, a water bath, a 1 mL pipette, a
vortex, solutions of Na-Acetate and KOH at room temperature, solutions of PNPP and acid
phosphatase on ice, a beaker with water and a stopwatch. First, organize your working area. For
instance, if you are right-handed, you may dispose the pipettes and solutions on the right side of
your station, whereas you will keep the rack with tubes, the vortex and the water bath, on the
left and center-back of your station.
1. Prepare and label 6 test tubes, each containing 2.0 mL of 0.5 M KOH. Label them: 0, 2, 4,
6, 10 and 15 (corresponding to the point times of the assay).
CAUTION: This is a concentrated solution of a corrosive base that can irritate your skin
and eyes. Wear gloves and safety glasses. For more info, consult the MSDS.
2. Pipet into a 7th tube (tube R) the proper volumes of 1.0M sodium acetate buffer (pH 5.7)
5
4
, Laboratory Class 3: Enzymes 2026
and water corresponding to your workspace dataset as described in Table 2. Mix with the
vortex.
5
5
BCH 2333 Lab 3: Enzymes Study Guide with Complete Solution | 2026 Update
PROCEDURES
Several aspects of the kinetics discussed above will be studied in this lab session, using a
commercial acid phosphatase preparation from wheat germ. First of all, you will investigate the
time dependence of the enzyme catalysed reaction. Following measurement of the initial rates
of reaction with fixed amount of phosphatase and variable concentrations of substrate in the
presence and absence of an unknown inhibitor, Hanes plots will be constructed, and KM and Vmax
will be determined from these plots. The analysis of the effect of the inhibitor on the kinetic
parameters of the enzyme will allow you to identify the type of inhibitor that you have used.
EXPERIMENT #1: Rate of product formation
As pointed out in the discussion of initial velocity the acid phosphatase assay must be carried out
under conditions where the rate of product formation is constant over the incubation period.
This is critical because the assay is a fixed time assay, and the enzyme is subject to product
inhibition. In order to check the rate of product formation, a time curve will be constructed. Each
student will use different amounts of enzyme, thus with the results of the whole group, you will
be able to analyze the dependence of the enzyme activity on the enzyme concentration.
In this experiment, you will be handling a rack with assay tubes, a water bath, a 1 mL pipette, a
vortex, solutions of Na-Acetate and KOH at room temperature, solutions of PNPP and acid
phosphatase on ice, a beaker with water and a stopwatch. First, organize your working area. For
instance, if you are right-handed, you may dispose the pipettes and solutions on the right side of
your station, whereas you will keep the rack with tubes, the vortex and the water bath, on the
left and center-back of your station.
1. Prepare and label 6 test tubes, each containing 2.0 mL of 0.5 M KOH. Label them: 0, 2, 4,
6, 10 and 15 (corresponding to the point times of the assay).
CAUTION: This is a concentrated solution of a corrosive base that can irritate your skin
and eyes. Wear gloves and safety glasses. For more info, consult the MSDS.
2. Pipet into a 7th tube (tube R) the proper volumes of 1.0M sodium acetate buffer (pH 5.7)
5
4
, Laboratory Class 3: Enzymes 2026
and water corresponding to your workspace dataset as described in Table 2. Mix with the
vortex.
5
5
