Literature Review

​ Biuret Test
The biuret test is widely recognized as the most reliable of the protein color tests. The
method was as follows: equal volumes of protein solution and a concentrated solution of a
strong alkali (sodium hydroxide) have been infused together, then a very dilute solution of
cupric sulphate was gradually added to the reaction mixture till a purplish-violet or
pinkish-violet color established, confirms the formation of protein-like material (A Study of
Modifications of the Biuret Test). Several different compositions of biuret test solutions are
evaluated, and researches using the biuret method for assessing the protein content of milk,
cheese, and meat are provided, according to Reichardt and Eckert W. To measure the pure
protein and casein content of skimmed milk, as well as the protein content of whole milk,
biuret test solutions containing potassium hydroxide and such a detergent have been used.
The addition of hydrogen peroxide, extraction, or additional assessments using a copper-free,
zinc-containing biuret reagent reduced lactose, fat, and turbidity-related biuret procedure
faults. Besides which, factors impacting the detection of nitrogen in soya products using the
biuret and orange-G dye-binding methods are evaluated. The orange-G dye-binding
technique and a variation of the biuret method, wherein the protein extraction as well as color
development happen simultaneously in an alkaline copper tartrate solution (an ester organic
compound), were used to analyze the nitrogen content of soya protein. As the overall nitrogen
content of the solution increases, so does the amount of nitrogen extracted into solutions.
The biuret test can also be used to estimate wheat protein. Pinckney's method has
been tweaked. The sample was 0.5g and was shaken thoroughly for 10 minutes with 1ml of
tetrachloromethane, followed by 50ml of reagent made by gradually stirring 40ml of 4
percent CuSO4.5H2O (Copper sulfate pentahydrate) into a mixture of 930ml water, 10ml 10
(Potassium hydroxide), and 20ml of 25% sodium potassium tartrate. The portion of the
solution was agitated until it was clear after it had been blended. This approach, which was
also easier and more accurate, yielded superior protein extraction. A quantitative biuret test
for milk serum proteins (Johnson, B. C. and Swanson, A. M.) is another enthralling notion
regarding this test. Caseinogen is a soluble milk protein that is transformed to insoluble
casein towards the presence of calcium ions. The caseinogen is precipitated at pH 4.6, and the
whey is concentrated by freezing, dialyzing, and lyophilizing. The biuret reaction is estimated
spectrophotometry using a 540 millimicron after the product is immersed in NaCl.
 ​ Lead Acetate/Sulfide Test

Lead acetate test is a test that detects amino acids that contain sulfur like cysteine and
cystine (Sapkota, 2020). Cysteine is a non-essential amino acid that is important in protein
production and other metabolic functions (University of Rochester Medical Center, n.d.).
Cysteine is the main protein in nails, skin and hair as it is important in production of collagen
in the body that affects the elasticity and texture. Furthermore, when two sulfur atoms of
cysteine are bonded to each other it will produce another amino acid called cystine
(Britannica, 2013). Cysteine is converted into cystine through the reduction process or the
addition of hydrogen molecules. In which, cystine is also detected in lead acetate test as it is
an sulfur-containing amino acid. Cystine is abundant in skeletal and connective tissues.
Thus, it is also particularly abundant in hair, horn, and wool (Britannica, 2018).

​ Hopkins-Cole Test
Hopkins-Cole test is a procedure that is also known as “glyoxylic acid test” since the
reagent contains a glyoxylic acid. This test was discovered by Frederick Gowland Hopkins
and Sydney W. Cole in 1901 while working on the discovery of tryptophan. Based on
Sapkota (2020), this test is very similar to the principle of Adamkiewicz reaction that also
detects the presence of tryptophan in the sample and forms a purple-color complex once the
tryptophan is detected. No formation of the purple-color complex will indicate that the
sample does not contain any presence of tryptophan. As for the limitation of this test, it is
important to not use any of the following in the experiment: nitrites, chlorates, nitrates, and
excess chlorides since these will prevent the reaction from occurring.

​ Precipitation of Proteins by Acids

Precipitation occurs due to the change in pH, altering interactions between proteins
and the watery environment. Through such binding with substances which are high in
Sodium Chlorides (NaCl) yielding to intramolecular interactions that can disrupt and
denature one specific protein. Proteins are similar to the actions of cations when in acid
substances, precipitated with the acid extremists to make insoluble salts (Righetti, n.d.)
Protein precipitation by acid, known as Heller’s test, discovered by Austrian Chemist -
Johann Florian Heller, a method relying on the changes of a solution pH. Detecting proteins
in one specific sample which denatures proteins by adding strong acids is its main principle.
This experimental test is usually performed to detect abnormal proteins with biological fluids
which represent pathological characteristics of such diseases. The most common proteins that
this test detects are albumin and globulin. Complex salts, used advantageously as protein
precipitants. With the works of some known people, this protein precipitation by acids is
commonly used in “conservatives” with the structure of protein+complex salts+third
component, optimum isoelectric point of the protein will be obtained and the desired pH from
the NaOH will be attained (Michael, n.d.)

​ Precipitation of Proteins by Heavy Metals

Proteins are usually at its optimum level or condition when it is at pH level of 4 to 7
since it is their isoelectric point. Moreso, continuing above that pH level will alter the
protein's condition into negatively charged; the positively charged metal ions neutralize the
charges of protein causing precipitation of the protein (ScienceDirect, 2011). Therefore,
precipitation of proteins by heavy metals is most effective at neutral to basic alkaline pH
value. Heavy metals such as silver, lead, mercury, etc. form a complex with the alkaline
proteins and precipitate, as the name suggests, these heavy metals are metals with high
atomic weight. According to Mona Sh AL-Harbi (2018), heavy metal will neutralize the
proteins by binding with the positive charge of the metal ion which will then cause the
protein’s precipitation as insoluble metal protein salt.
Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy
metal salt with a protein usually leads to an insoluble metal protein salt. This reaction is used

for its disinfectant properties in external applications. For example AgNO3 is used to prevent

gonorrhea infections in the eyes of newborn infants. Silver nitrate is also used in the
treatment of nose and throat infections, as well as to cauterize wounds.
“General consensus holds that proteins are the prime targets; heavy metals interfere
with the physiological activity of specific, particularly susceptible proteins, either by forming
a complex with functional side chain groups or by displacing essential metal ions in
metalloproteins. Recent studies have revealed an additional mode of metal action targeted at
proteins in a non-native state; certain heavy metals and metalloids have been found to inhibit
the in vitro refolding of chemically denatured proteins, to interfere with protein folding in
vivo and to cause aggregation of nascent proteins in living cells. Apparently, unfolded
proteins with motile backbone and side chains are considerably more prone to engage in
stable, pluridentate metal complexes than native proteins with their well-defined 3D
structure. By interfering with the folding process, heavy metal ions and metalloids profoundly
affect protein homeostasis and cell viability (Christen, 2014).”

Materials & Methods

​ Materials
Part A. Biuret test
​ Chemicals/Reagent
i. 2 ml gelatin solution
ii. 2 ml raw egg white solution
iii. small chicken strips
iv. 2 ml of milk
v. 1mL of 6M NaOH
vi. 1mL of CuSO4 .5H2O solution
​ Equipment
i. Test tubes
ii. Dropper
Part B. Lead Acetate/Sulfide test
​ Chemicals/Reagent
​ 2 ml gelatin solution
​ 2 ml raw egg white solution
​ small chicken strips
​ 2 ml of milk
​ 1ml of 6M NaOH
​ Lead acetate solution
​ Equipment
​ Test tubes
Part C. Hopkins-Cole test
​ Chemicals/Reagent
​ 2 ml gelatin solution
​ 2 ml raw egg white solution
​ Small chicken strips
​ 2 ml of milk
​ 1 mL Hopkins-Cole reagent
 ​ 1 ml concentrated H2SO4

​ Silver Nitrite (AgNO₃) solution

​ Equipment
​ Test Tubes
​ Dropper
Part D. Precipitation of Proteins by Acid
​ Chemicals/Reagent
i. 2 ml gelatin solution
ii. 2 ml raw egg white dissolve in 1M NaCl solution
iii. 2 ml of milk
iv. Concentrated Nitric Acid (HNO₃)
​ Equipment
i. Test Tubes
ii. Dropper
iii. Water Bath
Part E. Precipitation of Proteins by Heavy Metals
​ Chemicals/Reagent
​ 2 ml gelatin solution
​ 2 ml raw egg white solution
​ 2 ml of milk
​ 1M NaOH solution
​ Lead acetate solution
​ Silver Nitrite (AgNO₃) solution
​ Equipment
​ Test Tubes
​ Dropper
​ Methods

​ Biuret Test
​ Lead Acetate/Sulfide Test
​ Hopkins-Cole Test
​ Precipitation of Proteins by Acids
​ Precipitation of Proteins by Heavy Metals
Results & Discussion

​ Biuret Test
 ​ Expected/Theoretical Results

In the Biuret Test, the expected outcomes range from no color change (blue) to pink to
deep violet. Proteins are not present when there is no color change and thus the solution
remains blue. If the solution changes color from blue to violet (dark purple), peptides are
prevalent and if the solution changes color from blue to pink, peptides are indeed prevalent
also because peptides or peptones are short chains of amino acid residues. The biuret test is a
generic assay for peptide-bonded proteins or chemicals. The chemical biuret is made by
heating urea to 180° C. A purple-colored chemical is generated when the biuret is treated
with dilute copper sulfate in an alkaline environment. This is the foundation of the biuret test,
which is commonly used to identify proteins and amino acids. The copper (II) in the biuret
reagent bonds to the nitrogen atoms in the protein peptides to form a bond. The copper (II) is
now reduced and transformed into copper (I) because the deformation of peptide hydrogens
occurs in response to the reaction between copper (II) ions and nitrogen from peptide bonds
wherein as long as the environment is sufficiently alkaline.

A Biuret Test's composition includes hydrated copper sulphate, which provides the Cu
(II) ions that build the chelate complex. The chelate complex absorbs light at 540 nm, giving
it a violet appearance. As a result, a change in coloration from blue to violet shows the
presence of proteins. The color intensity increases as the number of peptide bonds increases.
The Biuret reagent is a solution composed of sodium hydroxide (NaOH) or potassium
hydroxide (KOH), hydrated copper (II) sulfate (Fehling’s solution), and potassium sodium
tartrate. Cu (II) ions are responsible for the reagent's distinctive blue color. Potassium
hydroxide solution provides the alkaline medium but does not participate in the reaction. The
chelate complex is stabilized by potassium sodium tartrate. It is stated in the expected result
that the gelatin solution, raw egg white solution, and the chicken strips results to positive,
which means proteins were present on the solution. However, for the experimental results it
showed a different outcome which leads to a negative result.

​ Experimental Results
Samples Observations

2ml gelatin solution Dark blue green color

2ml raw egg white Blue color

solution
 small chicken strips Blue color

2ml of milk Purple color

A Biuret test is a chemical test used to determine the presence of a peptide bond in a
substance. It is based on the biuret reaction in which a peptide structure containing at least
two peptide links produces a purple color when treated with alkaline copper sulfate. In
presence of an alkaline solution, blue-colored cu (II) or cupric ions create a chelate complex
of purple color, using oxygen of water and the unshared electron pairs of peptide nitrogen. On
the other hand, when the color turns blue or no color has changed, this results in a negative
result.

Shown in the table are the experimental results of the Biuret Test. There is only 1
solution that was tested positive and it is the milk. It is because milk detects the presence of
protein in the sample. In comparison to the researched theoretical results for this test, the
experimental results have shown distinct results for the appearance of the purple precipitate
or the copper sulphate. Based on the expected results, purple precipitate must be seen for the
sample such as in gelatin solution, raw eggwhite solution, and in the chicken strips. As shown
in the video, it was seen in other solutions that the person is putting a lot of reagent in the test
tube. On that note, the source of error for the Biuret test is that when adding an excess
amount of copper sulphate, it should be avoided because it will develop to a blue color
instead of purple color and that is why all the three first solutions resulted in negative.

​ Lead Acetate/Sulfide Test

​ Expected/Theoretical Results

Lead acetate test is a test that detects sulphur containing amino acids like cysteine,
cystine, and methionine (Basnet, 2020). The reagent used in this test is Lead acetate also
called Foli’s reagent (Karki, 2018). In which, the sulfur-containing amino acid reacts with
lead acetate or Foli’s reagent under alkaline conditions (through NaOH) to form a brown
precipitate. The S-H or S-S group in amino acids undergoes degradation in strongly alkaline
conditions. In which these sulfur-containing amino acids release sulfur at alkaline condition
and at high temperature. Thus, it will lead to the combining of sulfur with the alkali (NaOH)
that forms Na2, then it will further react with lead acetate that produces lead sulfide. This

lead sulfide will result in the formation of black precipitate or residue (Sapkota, 2020). These
black residues indicate the presence of sulfur-containing amino acid in the sample. That said,
the positive result of the lead acetate test is represented by the formation of black precipitate,
while the negative result is represented by the absence of the black residues.

In connection to the experiment, the gelatin will show no reaction as its amino acid is
not sulfur-containing. According to Rowles (2017) cooking collagen is what produces
gelatin. As stated in by De Pietro (2019), glycine, proline, and valine are the most common
amino acids in gelatin. Whereas these amino acids do not contain sulfur in its structure. On
the other hand, egg white, chicken strips, and milk is considered as a high-protein food
(Gunnars, 2020). These three samples contain all of the sulfur containing amino acids like
cysteine, methionine, and cystine. Hence, it will produce a positive result in the lead acetate
test.

​ Experimental Results

Table 2. Lead AcetateTest

Samples Observations

+ Lead acetate After water bath

2 ml gelatin solution A layer is formed: the upper Appearance of floating

part contains the melted sediments in the yellow
gelatin and the appearance colored solution.
of hazy white solution is
observed at the lower part.

2 ml raw egg white A layer is formed: a hazy Black precipitate is

solution white solution is observed observed.
at the upper part and a clear
solution is observed at the
bottom.

small chicken strips Solid chicken strips in a Black precipitate is

hazy white colored solution observed.
is observed
 2 ml of milk Hazy white colored solution Black precipitate is
is observed. observed.

Lead acetate test detects sulfur-containing amino acid samples whereas they produce
black residues or precipitate. Here, the experimental result is in accordance with the
theoretical result. The gelatin sample (Test tube A) has a negative result in the lead acetate
test because it does not contain amino acid with sulfur in its structure. It produces a yellow
colored solution with floating sediments in it as observed. Thus, the experimental result of the
raw egg white (Test tube B), chicken strips (Test tube C), and milk (Test tube D) also
confirms with the theoretical result. Black precipitate or residues are observed in their result.
That said, these three solutions have positive results for this test because the appearance of
black precipitate indicates the presence of sulfur-containing amino acid.

​ Hopkins-Cole Test
​ Expected/Theoretical Results

Hopkins-Cole test is a test that is done for detection of tryptophan-containing proteins in

the sample. The Hopkins-Cole reagent contains glyoxylic acid (C2H2O3) that reacts with

sulfuric acid. Through the use of a concentrated H2SO4, the tryptophan in the protein

solution will undergo the process of condensation with the aldehyde group on the glyoxylic
acid with the aid of the strong oxidizing sulfuric acid. For a positive result, it will form a
purple-color complex and for indication of a negative result, no purple-color complex will be
seen, this means that the sample has no tryptophan-containing proteins.
According to Picincu (2018), egg whites are a part of the egg that contains around
93% of protein and has all of the amino acids that are needed for the optimal functioning of
an individual’s body. Some of these are tryptophan, arginine, alanine, etc. Moreover, Webmd
(2020) also mentioned that chicken and milk have high amounts of tryptophan that shows
beneficial impact on mood depression, learning, memory skills, visual cognition, and
aggression control.
As an expected result, based from Sapkota (2020), the 2 ml gelatin solution will not
yield a positive result, thus, no purple-color complex will be seen as an indicator that this
sample does not contain a tryptophan. For the next test tubes which contain 2 ml of raw egg
white solution, small chicken strips, and 2 ml of milk, these will yield a positive result and
will form a purple-color complex in the test tubes implying that the samples are
tryptophan-containing proteins.
​ Experimental Results
Samples Observations

Addition of Hopkins-Cole reagent After the water bath

and H2SO4

2 ml gelatin solution White precipitate and visible No reaction

division of colors: green at the top
of the test tube and light green at
the bottom

2 ml raw egg white Appearance of white sediments at Appearance of purple-color

solution the bottom of the test tube complex

Small chicken strips Appearance of white sediments at Appearance of purple-color

the bottom of the test tube complex

2 ml of milk Appearance of white sediments at Appearance of purple-color

the bottom of the test tube complex

After the addition of Hopkins-Cole reagent and H2SO4 to the 2 ml gelatin solution(test

tube A), a white precipitate is seen and it is also visible that the color immediately divided
into two (2): green and light green. For test tubes B, C, and D, which contain 2 ml raw egg
solution, small chicken strips, and 2 ml of milk, respectively, only white precipitate appeared
at the bottom of the test tubes after the solutions were dropped.
In the next process, after the test tubes have been placed in the water bath for two (2)
minutes, the division of colors for test tube A becomes more visible. However, no
purple-color complex is seen in the test tube indicating that it yielded a negative result. Thus,
the gelatin solution does not contain any proteins. For test tubes B, C, and D, a purple-color
complex was seen at the bottom of the test tubes as a sign for a positive result. The theoretical
results and the experimental results for Hopkins-Cole Test yielded the same outcome.

​ Precipitation of Proteins by Acids

​ Expected/Theoretical Results
Acid precipitation, its main principle is to observe the changes of a solution’s
pH.
Each protein has a definite isoelectric point or the pl value. Small changes on its medium pH
can affect the protein structure alone. Thus, the addition of acids to the solution can lead to
the lowering of its pH and making the protein positively charged due to the reason that the
proton was captured by the amino groups.
Precipitation by acids, known as Heller’s test, can determine proteins in one
sample by adding strong acid (Sakopta, 2020). In an aqueous environment, the hydration
sphere which enlocoses proteins becomes disrupted due to the charges present. As for the
aforementioned statements, disruption may occur that can bring imbalance to the protein
structure which can lead to precipitation. Moreover, when the acid comes in contact with the
protein solution, white coagulated ring (precipitated protein) will be formed within a couple
of layers - positive result. On the other hand, absence of the white coagulated ring indicates
negative results.
​ Experimental Result
Samples Observations

Addition of Nitric Acid After the water bath

2mL of gelatin solution No reaction Gelatin dissolved, solution

is yellowish in color

2mL raw white egg Presence of bubbles at the Presence of white

dissolved in 1M NaCl milky white upper layer, precipitate
solution lower layer appeared
yellowish in color

2mL of milk No reaction

Shown in the table are the experimental results of the Precipitation of Proteins Acids
Test. For the first sample, 2 mL of gelatin solution, no reaction was observed upon the
addition of Nitric Acid. After the water bath, it was observed that the gelatin dissolved in the
solution and the sample exhibited a yellow color reaction.

​ Precipitation of Proteins by Heavy Metals

​ Expected/ Theoretical Results

Pauli, (1904) first assumed that the dialysed protein solution could not be precipitated
by the low concentration of heavy metal salt; whereas slightly alkalised, it became readily
precipitated. Isoelectric point is the pH at which protein is at its equilibrium or zwitterion
form (dipolar ion). They dissociate as acidic or basic depending upon pH of the solution; if
acid is added, base form is manifested and vice-versa. These properties are maximal at
isoelectric pH. Proteins can be precipitated out from its solution by a variety of substances
such as heavy metals. Such reactions which precipitate out protein from there solution are
called as precipitation reaction (Kanazariya, n.d.). Proteins are precipitated from their
solution by heavy metal ions. These metal ions precipitate the protein from their solution. On
the alkaline side of isoelectric pH, Protein dissociates as protein anion(Pr-) which combines
with positive metal ion (cation) to form insoluble precipitate of metal proteinate such as lead
albuminate and silver albuminate (Kanazariya, n.a.). Heavy metal ions precipitate proteins
from their solutions. The ions that are most commonly used for protein precipitation are
Zn2+, Fe3+, Cu2+, Sb3+, Ag+, Hg2+, Cd2+, and Pb2+. Among these metal ions, Hg2+,
Cd2+, and Pb2+ are known for their notorious toxicity. They can cause serious damage to
proteins (especially the enzymes) by denaturing them. This can result in death. The
precipitation occurs because proteins become cross-linked by heavy metals. Thus, lead
acetate has a chance to have error with the process of precipitation due to the denaturing
effect from its toxicity. Egg whites can be a sample representing albumin since it contains
albumin and is available to be used for precipitation reaction. Overall, the albumin sample
should react with the heavy metal ions to form precipitation of proteins, likewise, the gelatin
and milk are subjected to form the same result.

​ Experimental Result (wait di pa final)

Samples Observations

Lead Acetate Silver Nitrate

2ml gelatin solution No reaction Presence of precipitate

2ml raw egg white dissolve Formed 2 layers: top layer Presence of precipitate
in 1M NaOH solution consists of milky white
solution with bubbles while
bottom layer is clear

2ml of milk (casein dissolve No reaction Presence of precipitate

in 1M NaOH solution)

Heavy metal precipitation of proteins is based on the principle that metal ions
precipitate the protein from their respective solutions. Protein anion is formed when protein
dissociates, and it combines with a positive metal ion to form an insoluble precipitate. The
expected/theoretical results correspond with the experimental test results.

Each sample was treated with Lead Acetate for the first part of the experiment. The
gelatin solution showed no reaction and remained green in color, similar to its initial
appearance; the raw white egg formed two layers, one with a milky white solution at the top
and one with a clear solution at the bottom, similar to the color of the egg's albumen; bubbles
were also observed; and finally, the milk sample showed no reaction and is milky white in
color.

The second set involves the addition of Silver Nitrate to the samples, and all of them
formed precipitates, indicating that the positive charge of the metal ions has binded with the
negative charge of the carboxylate group in the proteins, resulting in the presence of
precipitates. Proteins in the samples are denatured as a result.

Answer to Questions:
1. In the Biuret test, write the chemical formula of this reaction and the resulting copper
complex. Do you think free amino acids will give a positive result with this reaction? why?
Garingo

2. What is the chemical reaction of protein to lead acetate test? What are the observable
changes that happen?

The SH group in amino acid of the protein will undergo degradation in strongly
alkaline conditions, then it will release sulfur at high temperature. Sulfur and NaOH will form
Na2 that will cause the formation of hazy white colored solution. With the addition of lead

acetate, the Na2 will further react with it and form lead sulfide. In which, the lead sulfide will
produce black residues.

3. In lead acetate test, which of the amino acids contain (-SH) group?
The amino acids that contain the (-SH) group are the cysteine, methionine, and cystine.

4. What is the principle of the Hopkins-Cole test?

The principle of the Hopkins-Cole test is about the formation of a purple-color complex
in the solution that is caused by the reaction of the glyoxylic acid and a tryptophan in the
sample as shown in the picture below.

With the use of this test, the presence of tryptophan will be detected. Moreover, with

the addition of H2SO4 to the solution, the protein solution is hydrolyzed and will help to

stabilize the glyoxylic acid and prevent decomposition and the release of CO2.

5. Why is albumin/egg white is dissolved in 1M NaCl in part D?

Protein precipitation caused by acids depends on the changes done to the pH level of the
solution. Proteins have their own pI value which means that any changes in their pH value
can cause disruption to the structure of that certain protein. The addition of acid causes
reduction in the protein’s pH value. In the case of Nitric Acid, it caused denaturation in the
Albumin hence why it dissolved in 1M NaCl.
6. By the end of the test in part D, did casein and gelatin coagulate? Are they still biologically
active? Why? - LALA
7. In part E, how can this technique help to eliminate poisoning by Pb²⁺ from water pipes or
accidental poisoning of Hg²⁺?
Drinking tap water can cause lead poisoning, especially if it comes from a lead pipe with
high acidity or low mineral content, which causes elevated blood lead levels in the body. In
contrast, accidental mercury poisoning can occur from consuming too much methylmercury
or organic mercury, both of which are found in seafood. The technique mentioned in part E
may aid in the elimination of Pb²⁺ poisoning from water pipes and/or accidental Hg2²⁺
poisoning through the consumption of proteins found in raw egg white and milk, as high
concentrations of these can be used as an antidote for poisoning. Because poison or heavy
metal ions act on the protein of raw egg white and milk rather than the protein sites of the
body, these forms precipitate with heavy metal salts and are eliminated from the body
through vomiting.
8. Why is it necessary to dissolve the egg white and casein in 1M NaOH in part E?
When an albumin solution is made more alkaline than its isoelectric point, it is charged
negatively owing to the following reaction:

If to such a solution the heavy metal salt is added the cationic metal ion will combine with
anionic protein ion, and when solution is not too dilute, the resultant compound, being less
soluble than either protein at isoelectric point or its alkaline salt, will be thrown out of
solution. The protein must be alkalised to some extent to be readily precipitated by heavy
metal salt. This fact suggests itself that the reaction of the medium plays an important role in
the precipitation of protein by heavy metal salt (Kodama, 1923). Sodium Hydroxide are
strong alkali that can induce the denaturation of proteins when added to albumin or protein
solution. In the presence of NaOH, the secondary structure of the denatured proteins was
destroyed, hydrogen bonds were broken, and the number of hydrophilic groups increased.
These phenomena enhanced the hydrophilic property of protein molecules; interaction
between these protein molecules resulted in the formation of a new aggregate (Ji, 2013).
Overall, it is inferred that sodium hydroxide is the activation/preparation for the proteins to
be readily precipitated when introduced with heavy metal ions.

Synthesis & Conclusion

Biuret Test
Biuret is a chemical made by heating urea to 180 degrees Celsius. The Biuret test is a
chemical test that uses the Biuret reagents, which include a 1% solution of Copper II sulphate
(CuSO4). The Cu2+ in the Biuret reagent binds to the peptide bonds in proteins, forming a
complex. As a result, this test aids in the recognition of peptide bonds in any substance.
Peptide bonds are formed when two acids are joined by a carbonyl and an amino group.
Furthermore, amino acids constitute the essential unit of protein, and they are linked together
by peptide bonds. This test is also known as Piotrowski's test, after a Polish physiologist
named Gustaw Piotrowski, who documented it in 1857. The biuret test is a simple method for
detecting the presence of proteins in bodily fluids. When Biuret is treated with dilute copper
sulphate in the presence of alkaline, a purple-colored material is produced. The creation of a
chelate complex, or copper coordination complex, is fundamental for its color.
Consequently, the color intensifies as the number of peptide bonds in a protein rises,
and we may identify peptide bonds in any biological fluid using this test's approach. In the
experiment, the first three solutions (gelatin solution, raw egg white solution, and chicken
strips) lead to negative results, because when the fehling’s solution was placed in the test
tube, no color was changed. For the second and third samples, they have turned negative as I
observed that the solution was more than enough. And for the last sample, which is milk, it
turned to a purple color which means protein and the copper sulphate was present in the
solution.
Lead Acetate/Sulfide Test
The second test is the lead acetate test that detects the presence of sulfur-containing
amino acid in a protein. The test is done through the addition of 1ml of 6M NaOH and 10-15
drops of lead acetate to the samples (2 ml gelatin solution, 2 ml raw egg white solution, small
chicken strips, 2 ml of milk). The gelatin solution shows negative results for the test while the
3 other solutions indicate positive results. Overall, the experimental result is in accordance
with the theoretical result. To connect this in real life, this test can be applied in health. The
detection of cystine in urine shows a possible disease as it is a pathological symptom of
cystine stones in kidneys and bladder.
Hopkins-Cole Test
The third test which is the Hopkins-Cole Test is a procedure that is done to detect the
presence of tryptophan-containing proteins. A white precipitate is observed after adding
Hopikins-Cole reagent and H2SO4 to the 2 ml gelatin solution (test tube A), as well as the
color splitting into two (2): green and light green. After dropping the solutions in test tubes B,
C, and D, which contained 2 ml raw egg solution, small chicken strips, and 2 ml milk,
respectively, only white precipitate emerged at the bottom of the test tubes. The division of
colors for test tube A becomes more obvious in the next step, after the test tubes have been
immersed in the water bath for two (2) minutes. However, there is no purple-colored complex
in the test tube, suggesting a negative result. As a result, there are no proteins in the gelatin
solution. A purple-colored complex was visible at the bottom of test tubes B, C, and D,
indicating a positive result. The Hopkins-Cole Test showed the same results in both
theoretical and experimental results.
This test can be applied in real-life especially in research because tryptophan is one of
the biggest contributions for normal growth of infants, this one is also responsible for the
maintenance and production of the body’s health, neurotransmitters, muscles, and enzymes.
When people know how to detect the presence of tryptophan, they can now make a decision
to include those foods rich in tryptophan such as milk, canned tuna, chicken, etc.
Precipitation of Proteins by Acids
Precipitation of Proteins by Acids is a test which is done to detect proteins in a given
sample by denaturation of proteins when a strong acid is added. The test uses Nitric Acid as a
reagent to denature the protein. The samples used are 2 mL gelatin solution, 2 mL raw white
egg dissolved in 1M NaCl solution, and 2 mL of milk placed in test tubes respectively. After
adding 5-10 drops of Nitric Acid, the samples are subjected to a water bath for a few minutes
to observe the results or reactions if there are any. Experimental results showed that
As for the real-life application, This test can be used biologically for detection of
proteins in biological fluids like urine. However, handling the Nitric Acid to be used in the
test should always be with extra care as it is corrosive. Considering that this type of test is
qualitative, it doesn’t measure the concentration of the protein present in a given sample.

Precipitation of Proteins by Heavy Metals

Proteins can be precipitated from their solutions using a technique known as
precipitation reaction (Kanazariya, n.d.). Heavy metals precipitate proteins by interacting
with the positive metal ion (cation) and the protein anion that binds to it as the protein
dissociates. The procedure was simple: two sets of three (3) test tubes were filled with 2 ml of
gelatin, raw egg solution, and milk solution. The first set received Lead Acetate, while the
second received Silver Nitrate.

The experimental results from the samples are consistent with the expected/theoretical
results, as the albumin sample, as well as the gelatin and milk, should react with heavy metal
ions. This is evident in the addition of Silver Nitrate, as protein precipitate formation was
observed in the given samples. To further explain the reaction, Heavy metals disrupt the
physiological activity of especially vulnerable proteins, resulting in the formation of a
complex or precipitate or the displacement of essential metal ions in metalloproteins.

Precipitation of proteins by heavy metals can be seen in common practices whenever a

person consumes poison or becomes poisoned as a result of heavy metals. Typically, people
who are poisoned, such as lead poisoning from contaminated tap water, are advised to
consume raw egg white to prevent lead from entering the body. Albumin, a protein found in
raw egg white, contains sulfhydryl groups in its structure that can bind to heavy metal ions.
Instead of the poison binding to protein sites within the body, these heavy metal salts will
bind with the albumin protein, forming a precipitate that will exit the body as the person
vomits. However, this treatment does not completely eliminate the absorbed poison; rather, it
only delays its absorption in the body, and any subsequent damage is irreversible.

References:
29 Experiment 4 Proteins. (n.d.). Studylib.net. Retrieved November 18, 2021, from
https://studylib.net/doc/8901956/29-experiment-4-proteins-objectives-1.-to-iso
late-the-cas%E2%80%A6
AL-Harbi, M. (n.d.). Protein. Faculty KSU EDU.
https://faculty.ksu.edu.sa/sites/default/files/lab2.pdf
Amino Acids in Egg Whites. (n.d.). Healthy Eating. Retrieved November 15, 2021,
from https://healthyeating.sfgate.com/amino-acids-egg-whites-2688.html
Andriyanto, A., Margarita, M., Mustika, A., & Sutardi, L. (2017). DUCK
EGGWHITE POTENTIAL IN TREATING SUBACUTE LEAD
POISONING. Asian Journal of Pharmaceutical and Clinical Research,
98–101. https://doi.org/10.22159/ajpcr.2017.v10s2.19499
Barlow, O., Biskind, M., & Sollmann, T. (1927). EGGS AND MILK AS
ANTIDOTES AGAINST MERCURIC CHLORIDE. Journal of the American
Medical Association, 88(9), 623–626.
https://doi.org/10.1001/jama.1927.02680350007003
Biuret Test. (n. d.). Biuret Test.
https://www.scribd.com/embeds/237999020/content?start_page=1&view_mod
e=scroll&access_key=key-fFexxf7r1bzEfWu3HKwf
Biuret Test - Principle, Preparation and Procedure. (n.d.). Www.vedantu.com.
https://www.vedantu.com/chemistry/biuret-test
Biuret Test for Protein. (2016). Brilliant Biology Student.
http://brilliantbiologystudent.weebly.com/biuret-test-for-protein.html
Biuret test for Protein. (2019). Revisionworld.com.
https://revisionworld.com/gcse-revision/applied-science/aqa-additional-applie
d-science/unit-2-exam-topics/food-science/food-tests/biuret-test-protein
Cherney, K. (2018, December 13). Understanding Mercury Poisoning. Healthline
Media. https://www.healthline.com/health/mercury-poisoning
Christen, P., Ibstedt, S., Jacobson, T., Sharma, S., & Tamás, M. (2014). Heavy Metals
and Metalloids As a Cause for Protein Misfolding and Aggregation.
Biomolecules, 4(1), 252–267. https://doi.org/10.3390/biom4010252
Cysteine. (n.d.). Www.britannica.com. Retrieved November 17, 2021, from
https://www.britannica.com/science/cysteine
Cysteine. (n.d.). Www.urmc.rochester.edu.
https://www.urmc.rochester.edu/encyclopedia/content.aspx?contenttypeid=19
&contentid=Cysteine
Eckert, W., & Reichardt, W. (1991). The determination of protein content of milk,
cheese, and meat with the use of the biuret reaction. Die Nahrung, 35(7),
731–738. https://doi.org/10.1002/food.19910350716
Isoelectric Point. (2011). Sciencedirect.com.
https://www.sciencedirect.com/topics/nursing-and-health-professions/isoelectr
ic-point
Janairo, G., Leonisa, S., Linley, M., Llanos-Lazaro, N., & Robles, J. (n.d.).
Determination of the Sensitivity Range of Biuret Test for Undergraduate
Biochemistry Experiments. http://e-jst.teiath.gr/issues/issue_23/Janairo_23.pdf
Johnson, B. C., & Swanson, A. M. (1952). Milk Serum Proteins. I. A Quantitative
Biuret Test for Milk Serum Proteins. Journal of Dairy Science, 35(10),
823–828. https://doi.org/10.3168/jds.s0022-0302(52)93762-4
Kanazariya, S. (n.d.). Precipitation Reaction of Protein.
https://www.zmchdahod.org/pdf/college/Reactions_of_Protein-01-11-2018.pdf
Lead in Tap Water & Household Plumbing: Parent FAQs. (2019).
HealthyChildren.org.
https://www.healthychildren.org/English/safety-prevention/at-home/Pages/Lea
d-in-Tap-Water-Household-Plumbing.aspx
Lead Poisoning. (2018). Healthline.
https://www.healthline.com/health/lead-poisoning
Lead poisoning. (2019). Nhsinform.scot.
https://www.nhsinform.scot/illnesses-and-conditions/infections-and-poisoning/
lead-poisoning
Michael, S. E. (1939). The precipitation of proteins with complex salts. Biochemical
Journal, 33(6), 924–930. https://doi.org/10.1042/bj0330924
Ophardt, C. (2019). Denaturation Protein. Elmhurst.edu.
http://chemistry.elmhurst.edu/vchembook/568denaturation.html
Pomeranz, Y. (1965). Evaluation of Factors Affecting the Determination of Nitrogen
in Soya Products by the Biuret and Orange-G Dye-Binding Methods. Journal
of Food Science, 30(2), 307–311.
https://doi.org/10.1111/j.1365-2621.1965.tb00307.x
Precipitation of Proteins. (n.d). Taif University.
http://www.magdyelnashar.com/new/images/pdf2/Practical%20Proteins%20Pr
ecipitation.pdf
Righetti, P. G., & Boschetti, E. (2013). Detailed Methodologies and Protocols.
Low-Abundance Proteome Discovery, 263–319.
https://doi.org/10.1016/b978-0-12-401734-4.00008-7
Sapkota, A. (2020, November 23). Heller’s Test- Definition, Principle, Procedure,
Result, Uses. Microbe Notes. https://microbenotes.com/hellers-test/
Sapkota, A. (2020, November 23). Hopkin’s Cole Test- Definition, Principle,
Procedure, Result, Uses. Microbe Notes.
https://microbenotes.com/hopkins-cole-test/
 Sapkota, A. (2020, November 23). Lead Sulfide Test- Definition, Principle,
Procedure, Result, Uses. Microbe Notes.
https://microbenotes.com/lead-sulfide-test/
US EPA. (2018, March 23). Basic Information about Lead in Drinking Water. US
EPA.
https://www.epa.gov/ground-water-and-drinking-water/basic-information-abo
ut-lead-drinking-water
Zhao, Y. (2014). Effects of Alkaline concentration, temperature, and Additives on the
Strength of alkaline-induced Egg White Gel. Poultry Science, 93(10),
2628–2635. https://doi.org/10.3382/ps.2013-03596
https://books.google.com.ph/books?hl=en&lr=&id=ztDqAAAAMAAJ&oi=fnd&pg=PA7&d
q=related+studies+in+biuret+test&ots=mdAhO3xPkH&sig=HiNWloOCcefJ_rB14XB7Rwf
XPno&redir_esc=y#v=onepage&q=related%20studies%20in%20biuret%20test&f=true