Part A Fractionation of egg white

Result

Test Observation

0.2 mL ovomucin + 1 mL water Cannot dissolve in water

0.2 mL ovalbumin + 1 mL water Dissolve completely in water

Table 1

Figure 1

Part B Denaturation of ovalbumin

Test Observation

Ovalbumin + Water The solution becomes cloudy

Ovalbumin + Urea The solution becomes colourless

Table 2
Figure 2

Part C Approximate isoelectric point of serum proteins

Table 3.1 Approximate isoelectric point of serum proteins

Tube no. pH Degree of turbidity

zero time after 15 min after 30 min after 45 min

1 4.1 - - - -

2 4.4 + + + +

3 4.7 ++ +++ +++ ++++

4 5.1 +++ +++ ++++ ++++

5 5.4 +++ ++++ ++++ ++++

 6 5.7 ++ +++ +++ ++++

7 6.0 ++ ++ +++ +++

8 6.3 + + ++ ++

Part D Denaturation of an enzyme

Test Observation

Rennin + milk Remain unchanged

Boiled rennin + milk Remain unchanged

(Rennin + urea) + milk Remain unchanged

(Rennin after pH adjustment) + milk HCl – Coagulated

NaOH – Remain unchanged

Conclusion
From the experiment, we can conclude that there are several properties of proteins such as
denaturation, coagulation and isoelectric pH. Denaturation is defined as partial or complete
unfolding of the native (natural) conformation of the polypeptide. This is usually caused by heat,
acids, alkalies, alcohol, acetone, urea, beta- mercaptoethanol.

Next, when proteins are denatured by heat, they form insoluble aggregates known as coagulum.
All the proteins are not heat coagulable, only a few like the albumins, globulins are heat
coagulable. In this experiment, when HCL is added into milk, the pH of milk was dropped below
the milk's normal pH. This affects the stability of casein molecules. Casein molecules undergoes
denaturation, forming soft gel at low pH then coagulating and separate out from watery portion.
Rennin is a type of enzyme, so it only function well at most suitable pH. Its optimum pH is pH
3-4.
Besides that, the pH at which a protein has equal number of positive and negative charges is
known as isoelectric pH.

Questions and Discussion

There are many types of globular proteins. The common protein was albumins which
soluble in water after hydrolysis with ammonium sulfate solution. The second protein was
globulins which partially soluble in water but dissolved completely in dilute salt solution. In the
process of preparing ovalbumin stock solution, saturated ammonium sulfate solution was used
repeated. Saturated ammonium sulfate solution is a salt solution which has a high solubility in
water which lowers the solubility of proteins. By adding ammonium sulfate to egg white, the
high concentration of salt will causes the cell wall to break thus releasing the proteins inside.
After centrifugation, supernatant containing proteins was taken and the pellet was discarded.
Further adding of saturated ammonium sulfate solution has lowered the solubility of proteins
causing the proteins to form precipitate. Phosphate buffer saline, PBS was used to dissolve the
precipitate formed by proteins and to store the proteins within the solution. PBS also used to
maintain the pH of proteins. In the process of preparing ovomucin stock solution, the
precipitation after adding water to egg white was ovomucin. After centrifugation, the ovomucin
pellet was dissolved by adding 3 drops of 2M NaCl but not dissolved in water. Excess drops of
NaCl will cause solution to from precipitate again. (Nelson and Cox, 2000; Biologicalworld.com,
2009) By observation, ovalbumin and ovomucin are globular proteins. Ovalbumin is an albumins
protein while ovomucin is a globulins protein.
Ovalbumin, for example, is the protein present in egg white ( 368 amino acids ), which is
present 70% in egg white . The sequence includes six cysteines with a single disulfide bond
between Cys74 and Cys121. The amino terminus of the protein is acetylated. Ovalbumin does
not have a classical N-terminal leader sequence, although it is a secretory protein. Instead, the
hydrophobic sequence may act as an internal signal sequence involved in transmembrane
location. This protein is neutral and soluble in water. Ovalbumin is rich in glutamate and
aspartate amino acids. It has a tertiary structure and has a hydrophobic core in centre of the
spherical shape (soluble in aqueous). The structure is small in length and width and therefore,
posses ovoid or spherical shape. Ovalbumin proteins are more complex in conformation than
collagen proteins. (“Structure And Function Of Ovalbumin,” n.d.)
Part B

Explain your observations in this section. How does urea act as a protein denaturant?

Urea acts primarily by disrupting the hydrophobic interactions that produce the stable core of
globular proteins. Urea can also disrupt hydrogen bond. The hydrogen bond polarized the
peptide groups. Intermolecular bonds and interactions weaken the overall secondary and tertiary
structure. The denaturation process occurs once urea access the hydrophobic inner core of the
protein. (Sciencing. 2018)

Part C

What is the approximate isoelectric point of the serum protein?

In Part C experiment, the approximate isoelectric point of the serum protein is pH 5.4 based on
the result. This is because the greatest degree of turbidity is at pH 5.4. The degree of turbidity
indicates that the amount of precipitate formed; in other word the serum protein has the lowest
solubility within this pH value. According to Malhotra, proteins tend to be least soluble at their
isoelectric point, therefore the precipitation of protein is at the maximum point (Malhotra, 2003).

The isoelectric point is the pH at which the amino acid does not migrate in an electric field, at the
same time the net charge is zero and it can be neutral at the certain pH value. Besides zero
charge, protein can have a net positive or negative charged, it is based on the pH condition and
the amino acid composition. In the acidic pH range, the proteins have a net positive. In the basic
pH range, the proteins have a net negative charge. The net charge of the protein molecules can
also be affected by the Ph, as well as the electrostatic repulsion between the protein molecules
(David, 1998). The solubility of the serum protein in its isoelectric point tends to be at minimum
and protein will aggregate when the net charge is zero (Hudson, 1992).
Part D
Rennin with milk under 37°C water bath had formed thicker consistency milk because a slight
coagulation of milk had formed, this is because rennin is optimum in body temperature of 37°C
(Panse and Arnold 2011). Boiled rennin with milk do not formed any coagulation because rennin
had been denatured during the boiling process, because treatment of rennin under high heat had
break the hydrogen and ionic bond that denatured the rennin. Rennin do not coagulate milk in
presence of urea because urea can disrupt the enzymatic activity of rennin on casein protein in
the milk. Urea can denature component in casein after the treatment of casein by rennin
(Yamauchi and Tsugo 1960). Rennin had formed coagulation of milk when treated with rennin
with presence of hydrochloric acid, it coagulated milk faster than the sample with only rennin
and milk because rennin works best in low pH, whereas at pH between 5 and 7, rennin will
coagulate milk slower than in low pH. Lastly with addition of rennin with sodium hydroxide in
milk, the milk did not have any changes because rennin cannot work in pH above 7 as sodium
hydroxide brought milk pH to alkaline (Cheeseman 1965).
Rennin normally occurred in stomach of young mammals, its inactive form, prorennin
synthesised by chief cells in stomach and activated into rennin by hydrochloric acid in stomach.
Its role is to coagulate milk so that milk precipitate will flow slower in gastrointestinal tract for
longer digestion. Casein in the milk has four major types, with alpha s1 and alpha s2, and beta
casein that are hydrophobic and will readily precipitated by calcium in the milk, and also kappa
casein that will self aggregated into micelles and keep alpha and beta casein from precipitated.
With presence of rennin, rennin will inactivates kappa casein and convert it into para-kappa-
casein, which will not form micelles anymore and therefore forming calcium-insoluble casein
precipitate (Bowen 1996).

Reference
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Hudson, B.J.F., 1992. Biochemistry of food proteins, 1st ed. UK: Elsevier Applied Science.

Malhotra, V.K., 2003. Practical biochemistry for students. New Delhi: Jaypee Bros. Medical
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Biologicalworld.com, 2009. Using PBS in Biochemical and Cell Biology Research.

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