Purpose
Objectives
Materials (for groups of two)
Procedure Background information should also include how proteins function as enzymes. The
students should be aware that enzymes function as catalysts and are specifically structured to
perform a certain chemical reaction. The natural function of esterase, which is the enzyme
used in this lab to hydrolyze pNPP, is to hydrolyze esters in the liver. Esterase is found in
the membranes of the liver cells like the endoplasmic reticulum. When the liver encounters
substances that the body cannot tolerate, it turns them into esters. The esters are then
hydrolyzed by the esterase and can be removed from the body easily.
If students have never used a colorimeter, describe how it measures the amount of light
passing through the mixture being tested. It will give the absorbance of light over the amount
of time. In this lab it will record the change of absorbance of the pNPP as it is hydrolyzed by
the esterase. The more pNPP is hydrolyzed, the darker yellow the solution will become.
The students must design an experiment to prove that esterase will hydrolyze pNPP,
changing it from clear to yellow using only the materials available. Also they should prove
that temperature and pH change will denature the esterase.
Preparation The liver homogenate can be prepared several months in advance. It should be prepared
from fresh liver and then frozen until ready to use. Slice about 100-200g of liver into
small pieces. Keep the liver cold at all times or the enzyme may become inactive. Place the
liver in a blender with one liter of distilled water. Blend it until no pieces are visible. Next,
centrifuge small amounts of the homogenate at 2000-5000 rpms for 10 minutes and extract
the liquid. Any solid liver may be thrown away. To prepare a "boiled" liver extract, follow
the same steps then boil the extract.
p-Nitrophenyl phosphate can be purchased from Sigma. Dissolve as directed (usually one
tablet in 20 ml of methanol).
The esterase buffer is prepared in three steps: Prepare the other buffers using purchased buffer pellets. Fischer Scientific or Sigma
Chemical are good sources.
Safety
Questions to Ask
Suggestions for Assessment
References and Resources
Where To Go From Here
Denaturing and Protein Function: Student Activity Background There are several functions which proteins can perform. They can be part of an
organism's structure. They can be used like hemoglobin in O2 transportation or used as
hormones such as insulin or growth hormone. Finally, they can be used as catalyst to speed
up chemical reactions in the form of enzymes. It is this last function which will be explored
in this lab exercise.
Esterase is an enzyme found in the liver of many animals including humans and cows.
The natural function of esterase is to hydrolyze (break down) esters in the liver. It is found
in the membranes of the liver cells, such as the endoplasmic reticulum. When the liver
handles toxins that the body can not tolerate and turns them into esters, the esters are then
hydrolyzed by the esterase enzyme and can be removed from the body easily.
Esterase will also hydrolyze a substance called p-Nitrophenyl phosphate (pNPP). When
this happens the pNPP will change from clear to yellow. The more pNPP is hydrolyzed the
darker yellow it will become. This change in color can be measured by the absorbance spectra
of a colorimeter.
Problem
Materials
Helpful Hints
Assessment
To provide students with an opportunity to apply their basic understanding of
proteins to
a problem involving the hydrolysis of a compound.
1) Improve students' skills in experimental design and analyzing data.
2) Gain skills in using a colorimeter as a tool to measure the hydrolysis of a compound.
Students should be familiar with the structure of proteins and how they can be
denatured. A
pre-lab activity might include cooking an egg and having students describe what they observe.
Afterward, talk about how proteins can be denatured by heat or change in pH.
Colorimeters can be purchased from: Vernier Software, 2920 S.W., 89th St.,
Portland, OR
97225, Phone: (503) 297-5317. Colorimeters cost approximately $99.00. The program used to
run the colorimeter and the interface box can also be purchased, as well; the program is called
"Data Logger."
1) Prepare Solution A by placing 130ml distilled water in a 250ml flask and adding 0.7g
20mM Potassium Phosphate Dibasic.
2) Prepare Solution B by placing 130ml distilled water in a 250ml flask and adding 0.54g
Potassium Phosphate monobasic (q.s. to 150 ml).
3) Prepare Solution C by pHing Solution A with Solution B to 7.4pH. Into another flask, add
0.2g Trition X-100. Pour Solution C into flask with the Trition. Add 0.074g EDTA then q.s.
solution to 200 ml. This can be stored at 4oC for several months.
Follow standard safety procedures for laboratory experiments (e.g., goggles, lab
coats or
aprons, etc.). Students should be warned to be careful when using acids, bases, and chemicals
(including pNPP) that can irritate their skin if they come in contact with them. If they should
spill some they should rinse throughly with water.
What is the purpose of proteins? How do enzymes function? What do you
expect the liver
and the boiled liver to do to the pNPP? What do you expect the different pH buffers to do to
the esterase?
The following can be used as assessment tools: 1) Student experimental design,
computer
data, and laboratory reports; and 2) Oral presentation of results and experimental design.
Acknowledgments to Nathan Mata and Carol Leibl for information used in this
lesson.
Students could do protein gels. For example they could take hemoglobin and
mix it with
detergents such as Era or Wisk that contain enzymes. Then run these proteins on an agarose
gel to see if Era or Wisk had the enzymes necessary to break down hemoglobin.
Teacher: Sue Lighthall; James Madison High School; San Antonio, TX
Research Host: Dr. Andrew Tsin; The University of Texas at San Antonio
Frontiers Summer Research Program 1996
Proteins are organic macromolecules composed of smaller subunits called amino
acids. The
number, type, and arrangement of the amino acids determine the shape of the protein which
directly controls the protein's function. The shape and function of a protein can be altered by
a process known as denaturing. Too high a temperature or change of pH can cause the
denaturing to take place. If this happens, then the protein can be rendered useless, unable to
perform it's normal job.
Design an experiment using the materials available and a colorimeter. The
experiment should
prove that cow's liver has esterase which will hydrolyze pNPP, changing it's color and
therefore it's absorbance. Also, prove that extreme temperature or pH will denature the
esterase.
1. Always have a source of liver, a buffer and the pNPP for every test.
2. Use 1000ul of buffer, 10ul of liver homogenate, and 50ul of buffer. Put them into the
cuvette in that order and do it very quickly. Also, keep the liver and esterase buffer on ice at
all times.
Oral presentation of results and a laboratory report.
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