Biochemistry Lab II

University of Houston BCHS 4311

Lab report

Part F: The Effect of Hydroxylamine, an Inhibitor of Peroxidase, on Reaction Rate

Hydroxylamine is toxic and may cause burns on the skin. Handle with care (gloves and

goggles). Place any waste containing hydroxylamine in the labeled waste containers

provided.

1. To test the effect of hydroxylamine, vary the concentration of 10% hydroxylamine

while holding the enzyme concentration and hydrogen peroxide concentration

constant. Use the volume of hydrogen peroxide and turnip slurry you chose as

your baseline in part B. You need to set up a reaction such that the inhibition rate

is 50% of the baseline rate in part B. This can only be determined by trial and error.

Use less hydroxylamine in the new trial if you got so much inhibition that the

reaction did not happen at all. Use more hydroxylamine if you do not get any

inhibition or see very little inhibition. You will need to choose an amount of

hydroxylamine between 50 μ and 500 μl. Let the hydroxylamine and turnip solution

stand for 1 minute after mixing by inverting the tube (covered with parafilm) before

adding H202. Use Table 9 below to set up your experiment.

Table 9a. Reagents that are required for investigating the effect of hydroxylamine on

enzyme activity. Step 1 above.

Tube Distille Guaicol Turnip 10% 1% Volume (Total =

d H2O (μl) extract Hydroxyl- H2O2 3 ml)
(μl) (μl) amine (uL)

Blank 1 (B1) 2184 30 30 120 60 3

Experimental 1 2184 30 30 120 60 3
(E1)
Blank 2 (B2) 910 30 30 50 60 3
Experimental 2 910 30 30 50 60 3
(E2)
Blank 3 (B3) 1820 30 30 100 60 3
Experimental 3 1820 30 30 100 60 3
(E3)
Blank 4 (B4) 3640 30 30 200 60 3
Experimental 4 3640 30 30 200 60 3
(E4)
Blank 5 (B5) 5460 30 30 300 60 3
Experimental 5 5460 30 30 300 60 3
(E5)
Blank 6 (B6) 7280 30 30 400 60 3
Experimental 6 7280 30 30 400 60 3
(E6)
Blank 7 (B7) 9100 30 30 500 60 3
Experimental 7 9100 30 30 500 60 3
(E7)
Table 9b. Absorbance readings for investigating the effect of hydroxylamine on enzyme

activity.

Absorbance at λ= 470 nm
Tube 20 s 40 s 60 s 80 s 100 s 120 s 140 s 160 s 180 s
E1 0.076 0.007 0.06 0.015 0 0 0 0.076 0.007
E2 0.18 0.051 0.161 0.111 0.067 0.009 0.014 0.18 0.051
E3 0.289 0.108 0.259 0.214 0.162 0.054 0.061 0.289 0.108
E4 0.361 0.147 0.335 0.297 0.237 0.097 0.096 0.361 0.147
E5 0.422 0.177 0.392 0.36 0.298 0.13 0.124 0.422 0.177
E6 0.475 0.203 0.437 0.41 0.341 0.157 0.147 0.475 0.203
E7 0.51 0.22 0.47 0.45 0.379 0.18 0.165 0.51 0.22

2. In order to create a Lineweaver-Burke plot, you will need to choose three

concentrations of hydroxylamine from your experiment above (choose the 50% inhibition

rate and then a concentration of hydroxylamine above and below the 50% point). For

each set concentration of hydroxylamine you select, hold the amount of turnip slurry

constant (use the same amount you used in step 1) and vary the amount of 1% hydrogen

peroxide (substrate) you use. Let the hydroxylamine and turnip solution stand for 1 minute

after mixing by inverting the tube (covered with parafilm) before adding H2O2. Use Table

10 below to record your methods.

Table 10. Reagents that are required for investing the effect of hydroxylamine on enzyme

activity. Step 2 above.

Tube Distille Guaicol Turnip 10% 1% Volume

d H2O (μl) extract Hydroxyl- H2O2 (Total = 3

(μl) (μl) amine (μl) ml)

Hydroxyl- Blank 1 (B1)

amine 2790 30 30 150 0 3

concentration 1
 Hydroxyl- Experimental 1

amine (E1) 2750 30 30 150 40 3

concentration 1

Hydroxyl- Blank 2 (B2)

amine 2790 30 30 150 0 3

concentration 1

Hydroxyl- Experimental 2

amine (E2) 2730 30 30 150 60 3

concentration 1

Hydroxyl- Blank 3 (B3)

amine 2790 30 30 150 0 3

concentration 1

Hydroxyl- Experimental 3

amine (E3) 2710 30 30 150 80 3

concentration 1

Hydroxyl- Blank 1 (B1)

amine 2760 30 30 140 40 3

concentration 2

Hydroxyl- Experimental 1

amine (E1) 2800 30 30 140 0 3

concentration 2

Hydroxyl- Blank 2 (B2)

amine 2740 30 30 140 60 3

concentration 2

Hydroxyl- Experimental 2

amine (E2) 2800 30 30 140 0 3

concentration 2

Hydroxyl- Blank 3 (B3)

amine 2740 30 30 140 80 3

concentration 2

Hydroxyl- Experimental 3

amine (E3) 2810 30 30 130 0 3

concentration 2

Hydroxyl- Blank 1 (B1) 2770 30 30 130 40 3

 amine

concentration 3

Hydroxyl- Experimental 1

amine (E1) 2810 30 30 130 0 3

concentration 3

Hydroxyl- Blank 2 (B2)

amine 2750 30 30 130 60 3

concentration 3

Hydroxyl- Experimental 2

amine (E2) 2810 30 30 130 0 3

concentration 3

Hydroxyl- Blank 3 (B3)

amine 2730 30 30 130 80 3

concentration 3

Hydroxyl- Experimental 3

amine (E3) 2760 30 30 140 40 3

concentration 3

Table 11. Absorbance readings over time at increasing H202 concentrations.

Abs orba nce λ= 470 nm ∆ A/min μmols [S] 1/v0 1/[S]

/min

Tube 20 40 s 60 s 80 s 100 s 120 s 140 s 160 s 180 s

1 0 0 0.014 0.04 0.058 0.076 0.09 0.102 0.115 0.165 12 0.004 26.3 250

2 0 0.00 0.022 0.04 0.059 0.076 0.088 0.099 0.109 0.165 12 0.004 27.8 250

3 0 0 0 0.00 0.021 0.037 0.052 0.066 0.073 0.331 24 0.008 41.7 125

4 0 0 0.01 0.03 0.053 0.072 0.089 0.102 0.118 0.159 12 0.004 25 250

3
 5 0 0.01 0.039 0.06 0.09 0.113 0.132 0.145 0.169 0.258 18 0.006 16.7 166.7

8 9

6 0.0 0.03 0.067 0.09 0.121 0.144 0.166 0.176 0.193 0.417 30 0.01 15.6 10

03 5 5

7 0 0 0 0.01 0.035 0.054 0.068 0.083 0.095 0.117 9 0.003 33.3 333.3

8 0 0.01 0.055 0.07 0.099 0.119 0.139 0.155 0.168 0.274 18 0.006 17.9 166.7

2 6

9 0 0.00 0.018 0.03 0.056 0.069 0.086 0.094 0.109 0.391 27 0.009 27.8 111.1

3 9

10 0 0 0.014 0.04 0.058 0.076 0.09 0.102 0.115 0.165 12 0.004 26.3 250

11 0 0.00 0.022 0.04 0.059 0.076 0.088 0.099 0.109 0.165 12 0.004 27.8 250

3. Dispose of any solutions containing hydroxylamine in a hydroxylamine waste container

(not the sink).

Data:

1. Plot a graph for hydroxylamine plus enzyme solution (absorbance vs time)

from step 2 (Table 11).

 Hydroxylamine plus enzyme solution (absorbance vs time)
0.25

1
0.2 2
Absorbtion (470 nm)

3
0.15 4
5
0.1 6
7

0.05 8
9

0 10
0 20 40 60 80 100 120 140 160 180 200 11
Time (s)

2. Calculate ∆ A/min for each assay from the selected experiment. Convert

these values to μmols/min as in Problem C.

∆c1 = 0.004 M/min = 0.004 (moles/L)/min

[0.004 (moles/L)/min] x 0.003 L= 12 x 10-6 moles/min

(12 x 10-6 moles/min)(1000 mmol/mole)(1000 μmol/mmol) = 12 μmol/min

∆c2 = 0.004 M/min = 0.004 (moles/L)/min

[0.004 (moles/L)/min] x 0.003 L= 12 x 10-6 moles/min

(12 x 10-6 moles/min)(1000 mmol/mole)(1000 μmol/mmol) = 12 μmol/min

∆c3 = 0.008 M/min = 0.008 (moles/L)/min

[0.008 (moles/L)/min] x 0.003 L= 24 x 10-6 moles/min

(24 x 10-6 moles/min)(1000 mmol/mole)(1000 μmol/mmol) = 24 μmol/min

∆c4 = 0.004 M/min = 0.004 (moles/L)/min

[0.004 (moles/L)/min] x 0.003 L= 12 x 10-6 moles/min

(12 x 10-6 moles/min)(1000 mmol/mole)(1000 μmol/mmol) = 12 μmol/min

∆c5 = 0.006 M/min = 0.006 (moles/L)/min

[0.006 (moles/L)/min] x 0.003 L= 18 x 10-6 moles/min

(18 x 10-6 moles/min)(1000 mmol/mole)(1000 μmol/mmol) = 18 μmol/min

∆c6 = 0.01 M/min = 0.01 (moles/L)/min

[0.01 (moles/L)/min] x 0.003 L= 30 x 10-6 moles/min

(30 x 10-6 moles/min)(1000 mmol/mole)(1000 μmol/mmol) = 30 μmol/min

∆c7 = 0.003 M/min = 0.003 (moles/L)/min

[0.003 (moles/L)/min] x 0.003 L= 9 x 10-6 moles/min

(9 x 10-6 moles/min)(1000 mmol/mole)(1000 μmol/mmol) = 9 μmol/min

∆c8 = 0.006 M/min = 0.006 (moles/L)/min

[0.006 (moles/L)/min] x 0.003 L= 18 x 10-6 moles/min

(18 x 10-6 moles/min)(1000 mmol/mole)(1000 μmol/mmol) = 18 μmol/min

∆c9 = 0.009 M/min = 0.009 (moles/L)/min

[0.009 (moles/L)/min] x 0.003 L= 27 x 10-6 moles/min

(27 x 10-6 moles/min)(1000 mmol/mole)(1000 μmol/mmol) = 27 μmol/min

∆c10 = 0.004 M/min = 0.004 (moles/L)/min

[0.004 (moles/L)/min] x 0.003 L= 12 x 10-6 moles/min

(12 x 10-6 moles/min)(1000 mmol/mole)(1000 μmol/mmol) = 12 μmol/min

∆c11 = 0.004 M/min = 0.004 (moles/L)/min

[0.004 (moles/L)/min] x 0.003 L= 12 x 10-6 moles/min

(12 x 10-6 moles/min)(1000 mmol/mole)(1000 μmol/mmol) = 12 μmol/min

3. Calculate the [S] as in Problem C.

A=εlc

c=A/εl

1 molar = A/(43.6 M-1cm-1 x 1 cm)

∆c = (∆A/min)/εl

∆c1 = (0.165/min)/[(43.6 M-1cm-1)(1 cm)] = 0.004 M/min

∆c2 = (0.165/min)/[(43.6 M-1cm-1)(1 cm)] = 0.004 M/min

∆c3 = (0.331/min)/[(43.6 M-1cm-1)(1 cm)] = 0.008 M/min

∆c4 = (0.159/min)/[(43.6 M-1cm-1)(1 cm)] = 0.004 M/min

∆c5 = (0.258/min)/[(43.6 M-1cm-1)(1 cm)] = 0.006 M/min

∆c6 = (0.417/min)/[(43.6 M-1cm-1)(1 cm)] = 0.01 M/min

 ∆c7 = (0.117/min)/[(43.6 M-1cm-1)(1 cm)] = 0.003 M/min

∆c8 = (0.274/min)/[(43.6 M-1cm-1)(1 cm)] = 0.006 M/min

∆c9 = (0.391/min)/[(43.6 M-1cm-1)(1 cm)] = 0.009 M/min

∆c10 = (0.165/min)/[(43.6 M-1cm-1)(1 cm)] = 0.004 M/min

∆c11 = (0.165/min)/[(43.6 M-1cm-1)(1 cm)] = 0.004 M/min

4. Create a Lineweaver-Burke plot. You will have 3 lines one for each

experimental condition in step 2, Part F.

Lineweaver-Burk Plot
45
1/Vo
40 y = 0.0433x + 4.2974
35
30 30 uL S

25 60 uL S
20 y = 0.0201x + 3.8813 100 uL S
15 Linear (30 uL S)
y = 0.02x + 3.1394
10
Linear (60 uL S)
5
Linear (100 uL S)
0
0 100 200 300 400 500 600 700 800 900
1/[S]

Discussion for Part F:

1. Explain the action of hydroxylamine on the enzyme's activity.

Enzyme activity increases with increasing concentration of hydroxylamine.

2. What are the KM and the Vmax with and without inhibitors?
KM is higher with an inhibitor than without it. The Vmax is unaffected both with and without

inhibitor.

3. What type of inhibition is this? Competitive, Uncompetitive, Mixed, Mixed

Noncompetitive?

This is the competitive type of inhibition. Hydroxylamine bears a structural resemblance

to a 1% hydrogen peroxide and thus competes with it for binding at the active site of an

enzyme. The hydroxylamine is not acted on by the enzyme but does prevent the hydrogen

peroxide from approaching the active site.