Final Lab Report
Buffer For New Enzyme Assay
3 Girls A Guy and a Biochem Lab
Leader: Laura Newswanger
Assistant Leader: Jamie Milchanowski
Chemical Specialist: Erin McDonald
Data Specialist: Will O’Brien

Objective
To find the most suitable substrate concentration, dilution, pH and temperature to combine with peroxidase that will result in the fastest and most stable of reaction.
Background
A buffer is a substance that resists large pH changes when small amounts of acids or bases are added. It is usually a substance that contains a weak acid and its conjugate base. Stabilizing pH levels is vital, therefore buffers are an essential part of life. For example, in humans, our blood has to stay at 7.4, but can vary between 7.35 and 7.45. If it falls below 7.35, acidosis will occur and if it rises above 7.45, alkalosis occurs. The optimal pH for most enzymes is between 6 and 8. Extremely acidic or basic environments cause enzymes to denature and therefore slow down the rate of reaction.
The concentration of a buffer is also important. The concentration is the sum of the concentration of weak acid and its conjugate base. The more molecules of buffer present, the more H+ and OH- ions can be absorbed without changing the pH; therefore, the greater the concentration of the buffer, the higher the buffering capacity. Buffers are most effective when there are equal amounts of weak acid and conjugate base. The most effective buffering occurs at one pH unit above and below its pKa.
Enzymes are proteins which act as catalysts in biochemical reactions. A catalyst cannot initiate a reaction that would not already happen on its own. However, it does drastically increase the rate of reaction in which a cell can carry out the reaction. Most enzymes are highly specific and tend to accelerate only one group of related reactions.
As a result, many different enzymes may be acting at the same time without interfering with each other. Hydrogen peroxide is a common enzyme. It is the end product of oxidative metabolism, and because it is a strong oxidizing agent it could become toxic if allowed to accumulate. To prevent toxic levels from occurring, eukaryotic cells have enclosed the enzymes producing peroxides within a membrane-bound organelle, the

peroxisome. Peroxisomes contain high concentrations of peroxidase, which reduces the harmfulness of peroxide by reducing it to water.
Peroxidase is an enzyme that can be used for treatment of industrial waste waters. Peroxidases can also be an alternative option of a number of harsh chemicals, eliminating harsh reaction conditions. There are many investigations about the use of peroxidase in many manufacturing processes like adhesives, computer chips, car parts, and linings of drums and cans.
An assay is used to detect the presence of an enzyme. Since enzymes are usually present in solutions in very small quantities, an assay must be designed to maximize sensitivity to detect the enzyme. If the assay is designed right it can be used to quantitate the amount of enzyme present in an unknown solution.
Materials
Rutabaga
Hydrogen Peroxide
Buffers
Enzyme dilution
Equipment
Spectrophotometer
Safety
Goggles will be worn at all times
Long hair will be tied back
Proper attire (solid shoes, pants, long sleeves) will be worn
When disposing of chemicals we would dilute everything down with water before pouring it down the drain.
Chemicals that were mixed with the carcinogenic dye were placed in a single container and not put down the drain to be disposed of correctly.

Procedure:
1.
Extract peroxidase from rutabaga a.
Peel, wash and cut rutabaga into 1” cubes. b.
Homogenize about 40g in 200mL of deionized water in a blender at high speed 3-4 times for 15 seconds. c.
Clarify the extract by suction filtration.
2.
Prepare Stock dilution:
To do this, start off by making a 1/1 dilution which contained 2.0 mL of enzyme extract and no distilled water. To make a 1/2 dilution, we put 2.0 mL of enzyme extract with 2.0 mL of distilled water. To make a 1/4 dilution, we took 2.0 mL of tube two and added 2.0 mL of distilled water. To make a 1/8 dilution, we took 2.0 mL of tube three and added 2.0 mL of distilled water. Lastly, to make a 1/16 dilution, we took 2.0 mL of tube four and added 2.0 mL of distilled water.
Tube #
1
2
3
4
5
Enzyme Added (mL)
2.0 mL of extract
2.0 mL of extract
2.0 mL of Tube #2
2.0 mL of Tube #3
2.0 mL of Tube #4 dH20 Added (mL)
0 mL
2.0
2.0
2.0
2.0
3.
Prepare Reaction Mixtures:
Test dilutions with the reaction mixture. To do this, make five reaction mixtures and a reagent blank. Put 4.0 mL of pH 7 buffer, 0.1 mL of dye, and 1.0 mL of hydrogen peroxide in each of the 6 tubes (exception: tube 6 had distilled water instead of hydrogen peroxide). Lastly, add 0.1 mL of the enzyme into each of the tubes, using the different dilutions. The reaction started after adding the enzyme. To test the reaction rates, read the absorbance at 30 seconds and one minute.
Tube # Buffer (mL) Dye (mL) H2O2 (mL) Enzyme
(mL)
1
2
4.0
4.0
0.10
0.10
1.0
1.0
0.10
0.10
3
4
5
6
4.0
4.0
4.0
4.0
0.10
0.10
0.10
0.10
1.0
1.0
1.0
1.0
0.10
0.10
0.10
0.10
From
Tube#
1
2
3
4
5
1
Dilution
1/1
1/2
1/4
1/8
1/16
1/1

4.
Prepare reaction mixtures for concentration of substrate.
To test the effect of substrate concentration on the rate of reaction, make seven reaction mixtures that differed in the amount of peroxide and distilled water. Each of the tubes contains 4.0 mL of pH 7 buffer, 0.1 mL of dye, and 0.1 mL of the enzyme and the best dilution. The amount of distilled water went as follows (from tube one to tube seven); 0.9 mL, 0.8 mL, 0.6 mL, 0.4 mL, 0.2 mL, 0 mL, 1.0 mL. The amount of peroxide went as follows (from tube one to tube seven); 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 0 mL. Again, add the enzyme last when ready for the reaction to occur. Read the absorbance at 30 seconds and one minute.
Tube#
1
2
3
4
5
6
7
Buffer (mL)
4.0
4.0
4.0
4.0
4.0
4.0
4.0
Dye (mL)
0.10
0.10
0.10
0.10
0.10
0.10
0.10 dH20 (mL)
0.90
0.80
0.60
0.40
0.20
---
1.0
H2O2 (mL) Enzyme (mL)
0.10 0.10
0.20
0.40
0.10
0.10
0.60
0.80
1.0
---
0.10
0.10
0.10
0.10
Tube #
1
2
3
4
5
6
5.
Prepare reaction mixtures for pH:
To test for the most suitable pH, prepare 6 test tubes. In each tube put 0.10 mL of dye, and 1.0 mL of H2O2. Put 4.0 mL of buffer (of different pHs) into each tube and add 0.10 mL of enzyme (that is properly diluted) just before measuring the absorbance. Place the tube into the spec20 and read the absorbance and 30 and 60 seconds.
Buffer (mL)
4.0 (pH 2)
4.0 (pH 4)
4.0 (pH 5)
4.0 (pH 6)
4.0 (pH 7)
4.0 (pH 8)
Dye (mL)
0.10
0.10
0.10
0.10
0.10
0.10
H2O2 (mL) Enzyme (mL) Dilution
1.0 0.10 1/4
1.0
1.0
0.10
0.10
1/4
1/4
1.0
1.0
1.0
0.10
0.10
0.10
1/4
1/4
1/4

Results
We did prior research and found that the best absorbency levels should all occur around .6 at about 60secs. The first graph and set of data show the enzyme concentration. The one that worked the best was a dilution of 1/4. In the second lab period, we found that 2/5 worked the best because it was a new set of enzyme product. The second data table and graph show the concentration of substrate. The best was Tube 5. The last graph and data shows the best pH level used with prior results from substrate and enzyme dilution. The best was a pH of 6 and 7. However, Lactic Acid and Acidic Acid showed results different from the others. It turned the solution green in the reaction instead of orange.

0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0
Dilution
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
0 100 200 300 400
Time (seconds)
500 600
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
Vo Vs. [S]
20 40
Time (seconds)
60 80
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
Tube 7

pH
2,5
2
1,5
1
0,5 pH 3.8 Lactic Acid pH 4.65 Acetic Acid pH 5.8 BIS TRIS pH 6.07 PIPES pH 7 NaPO4 pH 8.47 TRIS
0
0 100 200 300
Time (seconds)
Discussion/ Conclusion
Enzymes are extremely important in biochemical reactions. They are highly specific and each demonstrates its own characteristics. In this lab we examined the enzyme peroxidase while trying to make a stable reaction mixture assay. Proper dilution of the enzyme is important so that the rate of reaction can be easily and accurately detected. The concentration of the substrate is also very important. Substrates bind to the active site of an enzyme, forming an enzyme-substrate complex. Therefore, if there is enough concentration of substrate molecules, the binding sites should be saturated.
The initial rate of reaction will be at a maximum and will not increase even if the

substrate increases. Another characteristic concerning enzymes is pH. All enzymes have an optimum pH at which they are most active. There are many other factors that can affect how an enzyme will respond in a reaction. Such factors include temperature and light, as well as many others.
After the completion of this lab we were able to determine the standards for the most stable assay. We can now continue on to purify and isolate the enzyme to determine the molecules size, shape and chemical structure.

Reference Section
Green, Allen. "Buffers." Introduction to Biochemistry. HECO 224, Oneonta. 9 Sept.
2009. Lecture.
Helser, Terry. "Buffers and Titration". SUNY Oneonta. 9/15/2009
<http://employees.oneonta.edu/helsertl/Buffer.html>.
Mckee, Trudy. Biochemistry: The Molecular Basis of Life. Oxford: University Press,
2009.
Team Leader: Laura Newswanger
Assistant Leader: Jamie Milchanowski
Chemical Specialist: Erin McDonald
Data Specialist: Will O’Brien