2A analysis

In performing the xanthoproteic test, proteins when treated with concentrated HNO3 turn
yellow and change to orange when neutralized with NaOH. This is due to the nitration of
benzene ring(C6H5) prerent in the structure of such amino acids as tyrosine,tryptophan and
phenylalanine. The yellow stain on the skin produced by HNO3 is due to its reaction. The
sample yielded a positive result proved by the presence of a yellow precipitate. In Hopkins-
Cole Test, when protein mixed with glyoxylic acid is treated with concentrated H2SO4, a violet
ring is produced at the point of contact of the two solutions. This is due to the presence of an
indole nucleus in the trytophan component. The tryptophan condenses with the aldehyde to
form the colored compound. The sample did not yield violet rings in between the two liquids.
In Biuret test, alkaline solution of proteins treated with CuSO4 solution results in the
production of a rose-pink to violet, then purple color. This is due to the presence of peptide
linkage (--CONH2). All substances therefore containing this linkage respond positively to this
test. This test serves as a good index for determining the extent of protein hydrolysis. Because
the Cu ions in the biuret solution react with peptide bonds, a completely hydrolyzed protein
will have no peptide bonds and thus will display as negative. A peptide bond is a C-N bond
between a carboxylic acid group and an amine group. Amino acids lack a peptide bond which
is important in a positive Biuret test and will not yield a positive result.When ammonium
sulfate is used for salting out process, an excess of alkali should be added. In ninhydrin test,
when protein is boiled with ninhydrin (triketohydrindene hydrate) a blue color is produced.
This is due to the presence of the a-amino group in the molecule. All proteins, peptides and
amino acids except proline and hydroxyproline, give this typical reaction. IN the experiment, it
yielded a positive result. In Sakaguchi reaction, in alkaline solution, protein containing arginine
gives red color with a-naphthol and sodium hypochlorite. Since the sample contains sodium
hydroxide, the arginine present in egg white will give out a red color.

2A conclusion

Proteins present in egg white could be determined by using several tests, which give our
different colors taken as positive results in the presence of peptide bonds, specific amino acids
or whole proteins.. We have determined the principles involved in each test. Biuret test is a
key in explaining hydrolysis of proteins. We have determined that the presence of peptide
linkage (--CONH2) brings out the color change in the Hopkins-Cole test.

2B Analysis

1. Salt bridges result from neutralization of an acid and amine on the side chains. The final
interaction is ionic between the positive ammonium group and the negative acid group. Any
combination of the various acidic or amine amino acid side chains will have this effect.
Moreover, acids and bases disrupt salt bridges held together by ionic charges. A type of
double replacement reaction occurs wherein the positive and negative ions in the salt change
partners with positive and negative ions in the new acid or base added. Thus, it leads to the
formation of precipitate. Heavy metal salts usually contain Hg2+, Pb2+, Cd2+, and other
metals with high atomic weights. Since salts are ionic, they disrupt salt bridges in proteins.
The reaction of heavy metal salt with a protein usually leads to an insoluble metal protein salt.
In acidic environment, the positively charged protein molecules can react with the negatively
charged acid radicals of organic acids or alkaloidal reagents to form precipitate. Thus, these
proteins that precipitated are denatured. Practical use: TCA, Tungstic acid, are commonly used
for the preparation of protein-free filtrate of blood and other biological materials prior to
analysis of low constituents such as sugar, urea by specific methods. The most precipitation
occurs in the solution containing only ethanol since albumin is less soluble in ethanol. It is then
followed by the solution containing diluted HCl since albumin contains proteins that are soluble
in acids, thus, lesser precipitation occurs. The least precipitation occurs in the solution
containing alkaline solution since albumin has more solubility in the alkaline solution. Practical
use: a clinical application for the use of methanol as a protein precipitating agent is in the
estimation of bilirubin. Egg-white is faintly alkaline. Complete precipitation takes place in
faintly acidic solution the temperature at which coagulation takes place depends to a large
extent on the amount of acid and of salt present. Moreover, as temperature increases, the
rate of molecular movement within the solution is increased and the bonds within the proteins
begin to vibrate more violently and become disorganized. Coagulation occurs as protein
molecules then unfold and become entangled. Biuret test: excess of copper sulfate must be
avoided in making the Biuret test, since the color of the salt prevents the recognition of the
color produced in the reaction. The presence of ammonium salts interferes with the test. In
applying the reaction to solution containing these salts a large excess of sodium hydroxide
must be present. Compounds which give the biuret test must contain at least –CO-NH-groups.
The color formed in the reaction varies in shape with the complexity of the molecules.
Xanthoproteic test: the color is produced as a result of the formation of nitro-derivatives of the
compounds which contain a benzene ring, for example tyrosine Hopkins-Cole Test: the color
produced is due to the formation of a compound from the glyoxylic acid in the reagent and the
tryptophan in the protein. A similar color is produced when sulfuric acid is added to a protein
solution in the presence of a trace of formaldehyde.

2. Biuret solution is used to identify the presence of a protein. Biuret reagent is a blue solution
that when it reacts with protein, it will change to pink-purple.

Milk - Add 3 drops of Biuret Reagent Solution- Shake- Proteins will turn the solution
purple

2B Conclusion

There are many substances that could bring about the precipitation of proteins, be it inorganic
acids, alkaloidal, metallic, alcohol and even heat. There are also different tests to identify
proteins in the samples. Denaturation of proteins is one of the most useful processes for the
human body to obtain the necessary amino acids his or her body requires.

3A analysis

1. The Biuret test is a chemical test used for detecting the presence of peptide bonds. The
evidence for the test consists of formation of a violet-pink complex when cupric ion, in basic
solution, is added to any polymer such as protein which contains multiple amide bonds. A
blue-colored solution indicates fewer than two peptide bonds present or negative test. Thus,
simple amino acids do not give positive Biuret test because it does not contain multiple amide
bonds. Since the potato contains enzymes, it gives a positive Biuret test.

2. Catalase is a common enzyme found in nearly all living organisms exposed to oxygen.
It catalyzes the decomposition of hydrogen peroxide to water and oxygen. It is a very
important enzyme in reproductive reactions. Likewise, catalase has one of the highest turnover
numbers of all enzymes; one catalase molecule can convert millions of molecules of hydrogen
peroxide to water and oxygen each second.

Catalase is the one responsible for the decomposition of hydrogen peroxide into water and
oxygen.

2H2O2--------------- 2H2O + O2
catalase

A blue-green flame could be noticed when a matchstick is lit up and is drawn closer to the
mouth of the test tube. This indicates that oxygen has already escaped from the compound of
hydrogen peroxide.

3. Test tube 1 shows positive results by showing violet colored solution. Salivary amylase
breaks down starch to maltose. While in Test Tube 2, no reaction occurred since it contained
glycogen and salivary amylase is only specific in breaking down starch and not glycogen.
Some of the samples in the spot plate that were left open in the air reacted with the
atmosphere’s oxygen. Salivary amylase is specific for breaking down starch, while the enzyme
that could break down glycogen is called pancreatic amylase.

Amylase breaks starch down into sugar. Since the substrate must fit into the active site of the
enzyme before catalysis can occur, only properly designed molecules can serve as substrates
for a specific enzyme; in many cases, an enzyme will react with only one naturally occurring
molecule. The iodine serves as an indicator for the presence of starch. Iodine (I2) will reach
with iodide ion to produce the I3- ion. This ion will form a dark blue complex with the starch
molecule. The enzymes are very specific in their action and so is salivary amylase (enzyme) in
its action too. It basically breakdown carbohydrates from the food into simpler form for further
degradation but amylase do not breakdown carbohydrates to its simplest form.

3A conclusion

Biuret test is used to detect peptide bonds in a sample. Small chains of peptides and proteins
present in the sample give out a purple color, though amino acid residues that comprise
protein chains do not yield a positive result since they have no peptide bonds. Catalase plays a
very important role in breaking down hydrogen peroxide, which is a toxic substance found in
the body as a result of reactions. It is an extraordinary enzyme that saves us from free
radicals and immediate hydrogen peroxide poisoning. Salivary amylase is an enzyme that has
a specific role, and that is to break down only starch, into maltose. Glycogen cannot be broken
down by salivary amylase, but by pancreatic amylase.

3B analysis

1. When molecules collide, the kinetic energy of the molecules can be converted into chemical
potential energy of the molecules. If the chemical potential energy of the molecules become
great enough, theactivation energy of a exergonic reaction can be achieved and a change in
chemical state will result. Thus the greater the kinetic energy of the molecules in a system,
the greater is the resulting chemical potential energy when two molecules collide. As the
temperature of a system is increased it is possible that more molecules per unit time will reach
the activation energy. Thus the rate of the reaction may increase. In order to convert
substrate into product, enzymes must collide with and bind to the substrate at the active site.
Increasing the temperature of a system will increase the number of collisions of enzyne and
substrate per unit time. Thus, within limits, the rate of the reaction will increase. As the
temperatue of the system is increased, the internal energy of the molecules in the system will
increase. The internal energy of the molecules may include the translational energy,
vibrational energy and rotational energy of the molecules, the energy involved in chemical
bonding of the molecules as well as the energy involved in nonbonding interactions. Some of
this heat may be converted into chemical potential energy. If this chemical potential energy
increase is great enough some of the weak bonds that determine the three dimensional shape
of the active proteins many be broken. This could lead to a thermal denaturation of the
protein and thus inactivate the protein. Thus too much heat can cause the rate of an enzyme
catalyzed reaction to decrease because the enzyme or substrate becomes denatured and
inactive. 40 degrees Celsius is the optimum temperature, while in 60 degrees Celsius, the
enzyme is destroyed. In the experiment, the one placed in water with a temperature of 10
degrees Celsius will react slower than the one in the optimum temperature, 40 degrees
Celsius. Whereas the one put in 60 degrees Celsius will have its enzymes destroyed.

2. The pH of a solution can have several effects of the structure and activity of enzymes. For
example; pH can have an effect of the state of ionization of acidic or basic amino acids. Acidic
amino acids have carboxyl functional groups in their side chains. Basic amino acids have amine
functional groups in their side chains. If the state of ionization of amino acids in a protein is
altered then the ionic bonds that help to determine the 3-D shape of the protein can be
altered. This can lead to altered protein recognition or an enzyme might become inactive.
Changes in pH may not only affect the shape of an enzyme but it may also change the shape
or charge properties of the substrate so that either the substrate cannot bind to the active site
or it cannot undergo catalysis. In general enzymes have a pH optimum. However the optimum
is not the same for each enzyme. As of our results, when pepsin and pancreatin were added
with two different chemicals, their pH is altered and their properties were affected. The
optimum pH or pepsin is supposed to be at pH 2, but with the added HCl, it became pH 4, and
with sodium bicarbonate, it became 6, which slows its catalytic abilities. Meanwhile,
pancreatin, having an optimum pH of 9, is greatly affected by adding HCl, thus having the pH
of 5. Whereas sodium bicarbonate added to it slighty affects its pH and becomes 8. There will
be a pH, characteristic of each enzyme, at which the net charge on the molecule is zero. This
is called the isoelectric point (pI), at which the enzyme generally has minimum solubility in
aqueous solutions. In a similar manner to the effect on enzymes, the charge and charge
distribution on the substrate(s), product(s) and coenzymes (where applicable) will also be
affected by pH changes. Increasing hydrogen ion concentration will, additionally, increase the
successful competition of hydrogen ions for any metal cationic binding sites on the enzyme,
reducing the bound metal cation concentration. Decreasing hydrogen ion concentration, on the
other hand, leads to increasing hydroxyl ion concentration which compete against the
enzymes' ligands for divalent and trivalent cations causing their conversion to hydroxides and,
at high hydroxyl concentrations, their complete removal from the enzyme.

3B conclusion

Increase in rate would increase in temperature until a maximal rate is achieved. A region at
high temperature in which the rate decreases with increased temperature due to thermal
inactivation of the enzyme. There’s an optimal temperature, 40˚C at which the reaction is
most rapid. At 60 degrees Celsius, the enzyme is destroyed. Most enzymes have a
characteristic pH at which their activity is maximal. Above or low this pH, the activity declines.
Enzymes undergo denaturation which occurs at extremely high and low pH values. Enzymes
are affected by changes in pH. The most favorable pH value - the point where the enzyme is
most active - is known as the optimum pH.