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 Nutrition
RESULTS

A. Animal Dehydrogenase

Curdling Color Intensity

Milk
TT1 TT2 TT3 TT1 TT2 TT3
cow + + - ++ + +
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 H 2O2 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