Abstract

Proteins are very complex structures and have many variables that affect their function. This lab examined some of those variables. For the first part beef liver, which contains the enzyme catalase, was added to solutions of different concentrations of the substrate hydrogen peroxide. The second and third sections tested the effects of temperature and pH on cell permeability. The first part resulted with the enzyme being most effective at a substrate concentration of 4%.This is because at any concentration below this the enzyme is capable of breaking down more substrate than there is present. At any concentration above 4% the enzyme is bombarded with more substrate than it can breakdown and it cannot keep up its rate of reaction. The second section showed that the proteins function best at an optimal temperature and any temperature above or below this inhibit the function. The third section showed that the protein function decreased sequentially when the pH decreased. The second and third sections resulted the way they did because the transport and carrier proteins in the cell membrane became denatured because of the synthesized conditions. This allowed the betacyanin dye to leak out which created the color that was needed to determine the intensity and therefore the effect of the circumstances. The lab proved that substrate concentration, temperature and pH have significant effects on the function of complex proteins.

Introduction

Proteins are the most complex chemical structures in living organisms and are of utmost importance.[1] Their complexity comes from their specific three dimensional shapes that determine their function.[2] Proteins have many different functions including catalyzing biological reactions; these types of proteins are called enzymes.[3] Most enzymes are formulated to catalyze only one type of reaction, the ability of an enzyme to catalyze this reaction is dependent on its three dimensional structure.[4] An enzymes structure is affected by certain environmental circumstances, and is most efficient under optimal conditions.[5] One of these conditions is dependent on substrate concentration. A substrate is the reactant that an enzyme acts upon.[6] The concentration of a substrate will affect the rate at which the enzyme is able to catalyze. If the substrate concentration is steadily increased while the amount of enzyme stays the same the reaction speed will increase until it reaches a maximum.[7] This is a point where there is too much substrate and not enough enzymes to catalyze the reaction. Reactions cannot occur any faster than they do at this point.  An access of substrate however will not denature the protein. Another factor that affects the function of enzymes is temperature. The reaction rate increases along with temperature until the optimal temperature is reached after this the enzyme activity decreases quickly.[8] Temperature also affects cell permeability. In order for a living cell to survive it must have a functioning selectively permeable membrane.[9] If the membrane is distorted it can affect the structure of the proteins and the permeability.[10]Proteins are responsible for the transportation of nutrients in and out of the cell. If proteins are denatured they can no longer control what enters and exits the cell. When proteins are exposed to temperatures beyond those that they can withstand “the integrity of the cell membrane becomes compromised” and substances from the interior of the cell can escape, effectively denaturing and killing the protein.[11] pH levels have a similar effect on membrane proteins. The pH value is directly connected to membrane potential.[12] Every protein has an optimum pH, if the value is any higher or lower it is less effective until it reaches the point where it becomes denatured and is no longer effective at all.[13] At this point, like when denaturing occurs due to temperature, the membrane no longer is able to control what travels in and out of the cell. The purpose of this investigation was to investigate the realistic effects of increased substrate concentration, changes in temperature, and pH levels on the function of the complex protein structures.

Materials and Method

The Effect of Substrate Concentration on Enzyme Activity

A piece of beef liver was obtained and cut, with a scalpel, into 10 cubes approximately 1 cm x 1cm x 1cm. 4mL of 8% hydrogen peroxide was obtained and measured in a 10mL graduated cylinder. The hydrogen peroxide was then placed in a 100mL graduated cylinder. 2 drops of detergent were to the hydrogen peroxide and the cylinder was swirled. Forceps were then used to add one of the cubes of liver into the 100mL graduated cylinder with the hydrogen peroxide. After precisely 30 seconds the amount of foamed formed by the catalyzed substrate reaction in the 100mL graduated cylinder was recorded. The contents in the cylinder were then disposed of into the sink and the liver was placed in the garbage. The 100ml graduated cylinder was cleaned. The same procedure was repeated again with 8% hydrogen peroxide and the results were recorded. 4mL of 6% hydrogen peroxide was obtained and measured in a 10mL graduated cylinder. The hydrogen peroxide was then placed in a 100mL graduated cylinder. 2 drops of detergent were to the hydrogen peroxide and the cylinder was swirled .Forceps were then used to add one of the cubes of liver into the 100mL graduated cylinder with the hydrogen peroxide. After precisely 30 seconds the amount of foamed formed by the catalyzed substrate reaction in the 100mL graduated cylinder was recorded. The contents in the cylinder were then disposed of into the sink and the liver was placed in the garbage. The 100mL graduated cylinder was cleaned. The same procedure was repeated again with 6% hydrogen peroxide and the results were recorded. 4mL of 4% hydrogen peroxide was obtained and measured in a 10mL graduated cylinder. The hydrogen peroxide was then placed in a 100mL graduated cylinder. 2 drops of detergent were to the hydrogen peroxide and the cylinder was swirled. Forceps were then used to add one of the cubes of liver into the 100mL graduated cylinder with the hydrogen peroxide. After precisely 30 seconds the amount of foamed formed by the catalyzed substrate reaction in the 100mL graduated cylinder was recorded. The contents in the cylinder were then disposed of into the sink and the liver was placed in the garbage. The 100mL cylinder was cleaned. The same procedure was repeated again with 4% hydrogen peroxide and the results were recorded. 4mL of 2% hydrogen peroxide was obtained and measured in a 10mL graduated cylinder. The hydrogen peroxide was then placed in a 100mL graduated cylinder. 2 drops of detergent were to the hydrogen peroxide and the cylinder was swirled. Forceps were then used to add one of the cubes of liver into the 100mL graduated cylinder with the hydrogen peroxide. After precisely 30 seconds the amount of foamed formed by the catalyzed substrate reaction in the 100mL graduated cylinder was recorded. The contents in the cylinder were then disposed of into the sink and the liver was placed in the garbage. The cylinder was cleaned. The same procedure was repeated again with 2% hydrogen peroxide and the results were recorded. All material were cleaned and returned to their original places.

The Effect of Temperature on Cell Permeability

A beet was obtained and a corer was used to attain a beet cylinder which was cut into 12 even pieces approximately 0.5 cm thick with a scalpel. These 12 beet discs were placed in a watch glass and rinsed with water until the liquid they emitted was clear. A hot water bath was set up in a beaker which was half full of tap water and placed on a hot plate. Six test tubes were labeled 1-6 and a graduated cylinder was used to measure 10mL of water for each test tube. 2 beet discs were added to the 10mL of water in each test tube. Test tube 5 was placed in a fridge at 0°C and kept there overnight. Test tube 6 was placed in a freezer at -18°C and kept there overnight. Test tube 4 was kept in the rack at 23°C. The other 3 test tubes were placed in the hot water bath. A thermometer was placed in test tube 3 and the tube was removed when the temperature reached 40°C and placed in the rack. The thermometer was then placed in test tube 2 and the tube was removed when it reached 55°C and placed in the rack. The thermometer was then placed in test tube 1 and the tube was removed when the temperature reached 70°C and placed in the rack. Test tubes 1-4 were then observed and their color intensity was determined on a scale from 0 through 10 where 0 was clear and 10 was red, the results were recorded. Test tube 5 and 6 were observed approximately 24 hours later. The materials were cleaned and prepared to be reused for the next section.

The Effect of pH on Cell Permeability

A beet was obtained and a corer was used to attain a beet cylinder which was cut into 12 even pieces approximately 0.5 cm thick with a scalpel. These 12 beet discs were placed in a watch glass and rinsed with water until the liquid they emitted was clear. The test tubes were labeled 1-6 and a graduated cylinder was used to measure 5mL of pH 2 and the solution was placed into test tube 1. A graduated cylinder was used to measure 5mL of pH 3 and the solution was placed into test tube 2. A graduated cylinder was used to measure 5mL of pH 4 and the solution was placed into test tube 3. A graduated cylinder was used to measure 5mL of pH 5 and the solution was placed into test tube 4. A graduated cylinder was used to measure 5mL of pH 6 and the solution was placed into test tube 5. A graduated cylinder was used to measure 5ml of distilled water and the solution was placed in test tube 6. 2 beet discs were placed into each test tube. After approximately 2 minutes the solutions were observed and their color intensity was determined on a scale from 0 through 10 where 0 was clear and 10 was red, the results were recorded. All materials were cleaned and returned to their places. (This procedure was based on 2.2)[14]

Results

When the liver was added to the hydrogen peroxide an opaque white foam was formed and the volume increased over the 30 second period of time. The results of this test showed that the foam volume was the highest at a 4% concentration of substrate. The results from the two trials (Figure 1) are decisive enough to come to this conclusion. This is because when the results (Figure 1) are observed it can be seen that the volume of foam produced from 8% substrate is less than that produced from 2% but when put together all four percent concentration values do not correspond to sequential increases in foam volume. This shows that there is in fact a coloration between the amounts of substrate (in this case hydrogen peroxide) and enzyme (in this case catalase) function. This can be seen in Figure 1.2 where the results of the calculations of the rate reactions are shown. The percent concentration of 4% has a considerably faster reaction rate than the other three (2%, 6% and 8%). The reaction rate was calculated by subtracting the volume of the hydrogen peroxide and the detergent (5mL) from the average volume and dividing by time (30 seconds).

For the second part of the lab the test tubes were stored at different temperatures.  Upon temperature changes the solution changed color, the color intensities along with corresponding temperatures can be seen in Table 2. As the temperature value moved away from zero in both the negative and positive directions the color intensity increased, this can be seen graphically in Figure 2.

In the third part of the lab the beetroots were placed in different pHs. Upon submersion the solution changed color, the pHs along with corresponding color intensities can be seen in table 3. As the pH value increased and moved towards neutral the color intensity decreased. This can be seen graphically in Figure 3.

Discussion

When the liver is placed in hydrogen peroxide in theory it will dissociate into water and oxygen gas.[15] One way to prove that oxygen gas is in fact present is to conduct a glowing splint test; this involves lighting a splint and then extinguishing the flame only to the point where the end is glowing. This splint is then placed where the oxygen is believed to be present, if there is in fact oxygen the splint will reignite. This is because oxygen readily promotes combustion. The enzyme which catalyzed the reaction that created the oxygen appeared to work the most effectively at a concentration of 4% (Figure 1) and least effectively at 6%. This is because the 4% solution was the closet to the optimal concentration of substrate for the catalase enzyme. At 2% there was less substrate then the enzyme was capable of disassociating therefore the reaction was not completed to its full potential. At 6% and 8% there was too much substrate and the enzyme was bombarded and unable to keep up with the necessary reactions. Having the 8% solution create more foam volume then 6% was an unexpected result and the errors listed at below are most likely accountable for this anomaly. The enzyme catalase, which is found in the beef liver, is responsible for the breakdown of hydrogen peroxide. When hydrogen peroxide breaks down it forms water and oxygen gas. The oxygen and water reacted with the detergent that was added to the solution to produce the foam. The more effective and active the enzyme is the more decompositions of hydrogen peroxide it catalyzes and more oxygen is produced increasing the volume of foam. If the liver had been boiled before the experiment it would have been effectively denatured and the catalase would no longer have been able to carry out its metabolic function. When the liver was added to the hydrogen peroxide and detergent solution a reaction would not occur because it would be unable to breakdown the compound to water and oxygen and therefore foam would not form. Although this lab is effective in determining the efficiency of catalase under different substrate concentrations it does have several possible sources of error. First the detergent is added by means of a medicine dropper. This poses an error because the volume of each drop is not constant. If some of the drops were larger than others this creates more detergent for the oxygen to react with and in turn more foam creating the illusion that the enzyme is more effective at that specific substrate concentration than others. Also if there is less volume of detergent emitted the oxygen cannot for as much foam and the enzyme appears less effective. A second source of error is that the hydrogen peroxide was measured in a 10mL graduated cylinder and then transferred to a 100mL graduated cylinder, there is no way to ensure that all of the solution was transferred from one cylinder to another. It is inevitable that some of the hydrogen peroxide would have clung to the side of, and remained in the original cylinder; the amount that remained in each trial would have been different. Not having all of the hydrogen peroxide be transferred would result in less substrate for the enzyme to breakdown and a lower foam volume for the first two concentrations (2% and 4%) and a slightly higher foam volume for the second two concentrations (6% and 8%).A third source of error is that some pieces of liver may have had areas of fat inside them that were not visible and therefore there was less catalase in that piece of liver. This is very likely and if it were the case there would have been fewer enzymes to catalyze the reaction and this would have resulted in a smaller volume of foam causing an inaccurate result.

The second part of the lab showed that temperature had quite an effect on the permeability of beet cells. These cells contain a red dye called betacyanin. When the cell membrane works effectively the dye will not leak out of the cell. The lab showed that at room temperature the membrane remains effective but as the temperature traveled further away from 23°C ( both warmer and colder) the dye began to leak as the membrane failed. Increased or decreased temperatures will begin to denature the transport and carrier proteins in the cell membrane allowing the betacyanin to travel into the surrounding water. Freezing is a technique used in the preservation of food. By freezing food the function of enzymes is slowed considerably, freezing is also used for several other purposes. One example is the freezing of living tissue; this is done for several reasons including organ transplants. This is done to preserve tissues in the state they are in so that they are viable for the recipients.[16] Another example of freezing for preservation is the freezing of soil. This is done by construction companies to solidify the ground and eliminate any leakages.[17] A third example of freezing is that human reproductive cells can be frozen so that they may be used at a later date. This process has been used to create sperm and egg “banks” so people who may not have otherwise been able to can have children.[18] Freezing is very common in the food industry and although it may preserve some aspects it does not maintain others. For example, freezing preserves taste, shape and texture of the food but it will not preserve the proteins. In fact, as was learned in this lab, freezing temperatures actually denature proteins.[19] Also freezing does not preserve the fat in meat and over time it may go rancid and have a sour smell and taste.[20]Another problem with freezing food is that ice crystals can form in the food from the liquid that escaped through the denatured cell membrane. These ice crystals may be large and solid enough to damage the cell structures around them.[21] Temperature plays a key role in the efficiency of proteins.

The pH of the solution that the beetroot is placed in has a large effect on the permeability of the cells membrane. The cell leaked a considerable amount of betacyanin when placed in highly acidic solutions. The intensity of the color decreased as the pH approached neutral (Figure 3). This is because like changes in temperature, pH values that are not optimal for the protein will denature it causing it to not function and, in this case, allow betacyanin to leak through. Once source of error is that the color of intensity was judged on a scale of 0-10, the problem with this scale is that it is subjective and was not related back to a fixed sample. Each person sees the color intensity differently and therefore the results are not consistent or as reliable as they would be with a fixed source. A way to fix this error is to use a colorimeter.[22] A second source of error is in the ripeness of the beetroot. If the beetroot used in this lab was not ripe enough yet or too ripe the proteins may not have been as fully developed or beginning to die and the acid would have been able to denature them with ease therefore the color intensity would have been greater than it should have. A third source of error is that the betacyanin was more concentrated in some parts of the beetroot. This could be seen by just looking at the beetroot because some sections were darker than others. It is impossible to ensure that all of the beet discs have the same concentration of dye. Depending on how much betacyanin that was in the cell it would be able to emit more or less, changing the color intensity that the results were based on. pH also has a large impact on the function of proteins.

In summary, the purpose of this investigation was to examine the realistic effects of increased substrate concentration, changes in temperature, and pH levels on the function of the complex protein structures. The first section proved that an enzyme is more effective in lower concentrations of substrate. The second part showed that temperature has a large effect on the efficiency of proteins and the permeability of a cell membrane. The last section proved that a decrease in pH also denatures proteins and limits the effect of the membrane. In conclusion these three factors as well as many others have large effects on how proteins function.


[1] Imgrund, Ryan “Factors Involving Proteins”, 2009.

[2] Di Giuseppe, et al Maurice. Nelson Biology 12. South Melbourne: Nelson Thomson Learning, 2003.

[3] Di Giuseppe, et al Maurice. Nelson Biology 12. South Melbourne: Nelson Thomson Learning, 2003.

[4] Di Giuseppe, et all Maurice. Nelson Biology 12. South Melbourne: Nelson Thomson Learning, 2003.

[5] Di Giuseppe, et alMaurice. Nelson Biology 12. South Melbourne: Nelson Thomson Learning, 2003.

[6] Di Giuseppe, et al Maurice. Nelson Biology 12. South Melbourne: Nelson Thomson Learning, 2003.

[7] Arumugam, Karthik. “Enzymatic Treatment Of Fibers For Nonwovens.” (2005): 30. Web. 2 Oct 2009. <http://www.lib.ncsu.edu/theses/available/etd-08092005-123659/unrestricted/etd.pdf>.

[8] Arumugam, Karthik. “Enzymatic Treatment Of Fibers For Nonwovens.” (2005): 30. Web. 2 Oct 2009. <http://www.lib.ncsu.edu/theses/available/etd-08092005-123659/unrestricted/etd.pdf>.

[9] Imgrund, Ryan “Factors Involving Proteins”, 2009.

[10] Imgrund, Ryan “Factors Involving Proteins”, 2009.

[11] Gamper, Denice. “Investigating Factors That Affect Cell Membrane Permeability.” Summer Research Program for Science Teachers. 08 Aug 2009. Web. 3 Oct 2009. <http://www.scienceteacherprogram.org/biology/DGamper09.html>.

[12] Smithson Adair, Gilbert. “The Determination Of Membrane Potentials Of potentials Of Protein Solutions and the Valance of Protien Ions.” Low Temperature Research Station and the Biochemical Laboratory, (1934): n. pag. Web. 3 Oct 2009. <http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1253174&blobtype=pdf>.

[13] Kundrotas, Petras. “Optimum pH for protein-protein complexes..” (2006): n. pag. Web. 3 Oct 2009.

[14] Imgrund, Ryan “Factors Involving Proteins”, 2009

[15] Jones, Peter. “The Catalase-Hydrogen Peroxide System.” (1968): n. pag. Web. 6 Oct 2009.

[16] Fisher, Robyn. “Cold and cryo-preservation methods for human tissue slices.” Patent Storm (1994): n. pag. Web. 7 Oct 2009. <http://www.patentstorm.us/patents/5328821/fulltext.html>.

[17] Linde Group, The. “Freezing Soil.” Linde Industrial Gases. 2005. Web. 7 Oct 2009. <http://www.lindegas.com/international/web/lg/com/likelgcom30.nsf/DocByAlias/ind_soil>.

[18] Tucker, Michael J. “The Freezing of Human Oocytes (Eggs).” Georgia Reproductive Specialists (2007): n. pag. Web. 7 Oct 2009. <http://www.ivf.com/freezing.html>.

[19] Da-Wen, Sun. Handbook of Frozen Food Processing and Packaging. 1st. Boca Raton, FL: Taylor and Feancis Group, 2006. Print.

[20] Zotti, Ed. “Why is refreezing food bad? What exactly is freezer burn?.” (2005): n. pag. Web. 7 Oct 2009.

[21] Fellows, P.J. Food Processing Technology. 2nd. Boca Raton, FL: Wppdhead Publishing Limited, 2000. Print.

[22] “Colorimeter.” The Lab Depot. 2007. Red Clay Interactive, Web. 7 Oct 2009. <http://www.labdepotinc.com/c-454-colorimeter.php>.

Works Cited

Arumugam, Karthik. “Enzymatic Treatment Of Fibers For Nonwovens.” (2005): 30. Web. 2 Oct

2009. <http://www.lib.ncsu.edu/theses/available/etd-08092005-123659/unrestricted/etd

.pdf>

“Colorimeter.” The Lab Depot. 2007. Red Clay Interactive, Web. 7 Oct 2009.

<http://www.labdepotinc.com/c-454-colorimeter.php>.

Da-Wen, Sun. Handbook of Frozen Food Processing and Packaging. 1st. Boca Raton, FL:

Taylor and Feancis Group, 2006. Print.

Di Giuseppe, et al Maurice. Nelson Biology 12. South Melbourne: Nelson Thomson Learning,

2003.

Fellows, P.J. Food Processing Technology. 2nd. Boca Raton, FL: Wppdhead Publishing Limited,

2000. Print

Fisher, Robyn. “Cold and cryo-preservation methods for human tissue slices.” Patent Storm

(1994): n. pag. Web. 7 Oct 2009. <http://www.patentstorm.us/patents/5328821/fulltext.

html>.

Gamper, Denice. “Investigating Factors That Affect Cell Membrane Permeability.” Summer

Research Program for Science Teachers. 08 Aug 2009. Web. 3 Oct 2009.

<http://www.scienceteacherprogram.org/biology/DGamper09.html>.

Imgrund, Ryan “Factors Involving Proteins”, 2009.

Jones, Peter. “The Catalase-Hydrogen Peroxide System.” (1968): n. pag. Web. 6 Oct 2009.

Kundrotas, Petras. “Optimum pH for protein-protein complexes..” (2006): n. pag. Web. 3 Oct

2009.

Linde Group, The. “Freezing Soil.” Linde Industrial Gases. 2005. Web. 7 Oct 2009.

<http://www.lindegas.com/international/web/lg/com/likelgcom30.nsf/DocByAlias/ind_soil>.

Smithson Adair, Gilbert. “The Determination Of Membrane Potentials Of potentials Of Protein

Solutions and the Valance of Protien Ions.” Low Temperature Research Station and the Biochemical Laboratory, (1934): n. pag. Web. 3 Oct 2009. <http://www.pubmedcentral.

nih.gov/picrender.fcgi?artid=1253174&blobtype=pdf>.

Tucker, Michael J. “The Freezing of Human Oocytes (Eggs).” Georgia Reproductive Specialists

(2007): n. pag. Web. 7 Oct 2009. <http://www.ivf.com/freezing.html>.

Zotti, Ed. “Why is refreezing food bad? What exactly is freezer burn?.” (2005): n. pag. Web. 7

Oct 2009

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Robert Jerry
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thank you

ambrose
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nice article

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