Laboratory Conference

EXPERIMENT #2 – ENZYMES

Submitted by:

1st year section C; Group 2

Israel, Mara Aren C. _____________ ________________________

Jao, Dinky Mary-Del _____________ ________________________

Jimenez, Cheenee Carla R. _____________ ________________________

Kang, John Dainiel A. _____________ ________________________

Lecciones, Chantelle Joy R. _____________ ________________________

Submitted to: Department of Biochemistry and NutritionRESULTS

A. Animal Dehydrogenase

MilkCurdling Color Intensity

TT1 TT2 TT3 TT1 TT2 TT3cow + + - ++ + +goat + + - + ++ +

breast - - - + ++ +evaporated + + - + + +

carabao + + - ++ + +

Curdling: (+) with curdling; (-) without curdling

Color intensity: (++) intense coloration; (+) normal coloration

B. Animal Catalase

Liver Evolution of oxygen Presence of flame

Pork + +

Chicken + +

Goat + +

Cow + +

Fish + +

DISCUSSION

A. Animal Dehydrogenase

Methylene blue is used in the experiment to stain the milk and to check the amount of dissolved gasses present in the milk and presence of bacteria. Methylene blue will undergo reduction due to hydrogen donators present in the milk like aldehydes, citrates, succinates and a few more. The amount of dissolved gasses, like Oxygen compete with methylene blue in the

reduction since they also have affinity to the hydrogen present. Thus, the more oxygen in the milk, the more intense the blue coloration will be.

The addition of formaldehyde acts as a hydrogen donator w/c enhances the reduction of both methylene blue and oxygen present. Also it decreases bacterial contamination and prolong its keeping quality.

Boiling of the milk was done to create a force to let all dissolved gas rise to the surface of the milk but will not get released due to the fatty layer on the surface of the milk. The bubbles formed during boiling will not burst easily due to the difference in pressure where in the bubble created has less pressure inside compared to outside the bubble. Also, heating of the milk may cause death of the bacteria present and denaturation of enzymes present. In addition, increased temperature in milk increases enzyme activity and decreases the time for each reaction.

Paraffin was added to all samples to prevent gas exchange from the atmosphere and the milk.

Cow’s milk

In the experiment, we can see that cow milk had a very intense blue coloration in test tube 1, this may have been due to the fact that the milk in this test tube was boiled causing all the dissolved gasses to rise to the surface that when the formaldehyde was added to the mixture. The oxygen immediately reacted with the hydrogen being released by formaldehyde which caused less reduction of the methylene blue.

In the second test tube of cow’s milk, the color intensity was not that intense due to the dissolved gasses were spread throughout the mixture unlike in the first test tube where in the milk was boiled causing all the gas to rise. Since the intensity of dissolved gas was not that intense in the surface, the hydrogen released by the formaldehyde was able to react with most of the methylene blue which resulted in a less intense coloration.

For the third test tube of cow’s milk, the color intensity was similar with test tube 2 since it was not heated as well and methylene blue was allowed to react normally with the hydrogen released or present in the milk.

Goat’s milk

As we can see from the results, test tube 1 had a less intense coloration compared to test tube and test tube 3 had a similar intensity with test tube 1. This just shows that in test tube 2, there were less reactants that reacted to methylene blue to reduce it as compared to test tube 1 and 3 which had more reactants. This reactants may be the presence of hydrogen ions present in the milk, hydrogen ions given off by hydrogen donators like aldehydes.

Breast milk

Breast milk, is composed of proteins, fat, carbohydrates and other minerals. It also contains lactate dehydrogenase.

Methylene blue was exposed to a reducing agent, which was lactate dehydrogenase. Methylene blue acts as the hydrogen acceptor, and the oxygen from milk was removed. Methylene blue is substituted for NAD+, the blue color of methylene blue will result to a lighter blue color compared with the original as it is reduced and the lactic acid is oxidized to pyruvic acid.

The intensity of color in test tube 1 was less which is due to the fact that there was a loss of oxygen brought about by heating.

Evaporated milk

Evaporated milk is also known as dehydrated milk, wherein the sixty percent of the fresh milk was removed. There was curdling in test tube 1 and 2 and none in test tube 3, just like the other kinds of milk. The intensity of color was equal for the three test tubes. The amount of oxygen in the three test tubes may be in equal amount since the color of methylene blue was almost the same.

Carabao’s milk

It showed that the test taubes added with formaldehyde showed a short reduction or reaction time as evidenced by the slow scattering of methylene blue in the milk solution as compared to test tube 3 which showed almost complete diffusion of methylene blue. In the experiment, paraffin oil was added to avoid influence of atmospheric oxygen.

Putting the test tubes in a water bath increases the temperature thus increasing the enzymatic reaction. The maximum temperature at which heat catalyzes the activity is at 40°C. After which, the enzyme will undergo denaturation

Furthermore, curdling was seen in test tubes 1 and 2 of the cow’s, goat’s, carabao’s and evap milk. Curdling occurs when acidification of milk happens. Acidification of milk occurs when, lactose turns into lactic acid. This process is due to the presence of proton donators such as aldehydes.

In the Animal Dehydrogenase experiment, test tubes added with formaldehyde (cow, goat, evaporated, carabao milk) showed a short reduction as evidenced by the slow scattering of methylene blue in the milk solution as compared to test tube 3 (breast milk) which showed almost complete diffusion of methylene blue. Paraffin oil was also added to avoid influence of atmospheric oxygen. Putting the test tubes in a water bath increases the temperature thus increasing the enzymatic reaction.

B. Animal Catalase

Catalase is a common enzyme found in nearly all living organisms that are exposed to oxygen. It is a heme containing redox enzyme. It is found in high concentrations in a

compartment in cells called the peroxisome. Catalase is one of the most potent catalysts known. The reactions it catalyses are crucial to life. Catalase catalyses conversion of hydrogen peroxide (a powerful and potentially harmful oxidizing agent) to water and molecular oxygen. Catalase also uses Hydrogen Peroxide to oxidize toxins including phenols, formic acid, formaldehyde and acohols.

2H2O2    2H2O + O2

H2O2 is a powerful oxidizing agent and is potentially damaging to cells. By preventing excessive H2O2 build up Catalase allows important cellular processes which produce H2O2 as a byproduct to take place safely.

All known animals use this enzyme in every organ, with particularly high concentrations occurring in the liver. Its major function within cells is to prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of metabolic processes - primarily that of the electron transport pathway. This is because hydrogen peroxide is a powerful and potent harmful oxidizing: to prevent damage, it must be quickly converted into other, less dangerous substances. To this end, catalase is frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide into less reactive gaseous oxygen and water molecules.

One characteristic of catalase is that it has one of the highest turnover numbers of all enzymes. One molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second. This is why generation of bubbles in the animal catalase experiment was very quick upon the combination of the liver suspension and the hydrogen peroxide.

In this experiment, our source of enzymes is animal liver particularly in pork, chicken, goat, cow and fish and our substrate as hydrogen peroxide. Upon immersing the tube filled with hydrogen peroxide and animal liver in the beaker of water, you can see the oxygen gas bubbles escaping and causing the reaction to foam. This is because; Catalase breaks down hydrogen peroxide into water and oxygen. After the tube was released in the beaker, we immediately plunge splinter into the tube. It can be noted that there is a presence of a bright glow because of the oxygen present in the test tube.

Positive evolution of oxygen is observed as seen in the presence of bubbles and the flame created, thus the reaction: 2H2O2    2H2O + O2 is proven. Bubbles formed, thus we shall conclude that the organism’s liver is catalase-positive. Also, Oxygen itself is not a combustible substance. It reacts vigorously with combustible materials, especially in its pure state, releasing heat in the reaction process, thus causing the sudden spark on the glowing splinter

In clinical significance, Liver contains many enzymes, each important for detoxifying the body. One of the reasons breaking down hydrogen peroxide is important is because if left alone, hydrogen peroxide in the blood can produce free radicals. Free radicals can cause damage to different parts of the body. Catalase is also important because it can be used in textile industry by removing hydrogen peroxide from fabrics to make sure the material is peroxide-free.

CATALASE TEST

Some bacteria and macrophages can reduce diatomic oxygen to hydrogen peroxide or superoxide. Both of these molecules are toxic to bacteria. Some bacteria, however, possess a defense mechanism which can minimize the harm done by the two compounds. These resistant bacteria use two enzymes to catalyze the conversion of hydrogen peroxide and superoxide back into diatomic oxygen and water. One of these enzymes is catalase and its presence can be detected by a simple test. The catalase test involves adding hydrogen peroxide to a culture sample or agar slant. If the bacteria in question produce catalase, they will convert the hydrogen peroxide and oxygen gas will be evolved. The evolution of gas causes bubbles to form and is indicative of a positive test.

For differentiation of Streptococci (catalase-negative), Staphylococci (catalase-positive) and Listeria (catalase-positive) from beta hemolytic streptococci.

The enzyme catalase is present in most cytochrome-containing aerobic and facultative anaerobic bacteria. Organism lacking cytochrome system is indicative of the lack of catalase enzyme and are unable to break down hydrogen peroxide.

Catalase + :

-All species of genus Staphylococcus-Family Enterobacteriaceae (which includes Citrobacter, E.Coli, Enterobacter, Klebsiella, shigella, yersinia, proteus, salmonella, serratia)

REFERENCEs

Murray,R. K, Granner, D. K., Rodwell, V. W. (2006). Harper’s Illustrated Biochemistry 27th Edition. Singapore: McGraw-Hill Companies, Inc.

Fay, A. C. The detection of formaldehyde in milk by means of methylene blue reduction test. Retrieved from http://jds.fass.org/cgi/reprint/18 /5/327.pdf

Thorrnton, H.R. and Hastings, E. G. Studies on oxidation-reduction in milk: The methylene blue reduction test. Retrieved from http://jds.fass.org/cgi/reprint/13/3/221

Whitehead, H. R. The reduction of methylene blue in milk: The influence of light. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1254496/pdf/biochemj01126-0005.pdf

http://www.itisacqui.it/sitob/formagette/curdling.htm

http://www.biochemj.org/bj/032/0503/0320503.pdf