Nursing Biochemistry

Laboratory Report

Enzymes: Chemical Nature

and Specificity
Activity No. 9

Abain, Annrisa L.
Alibasa, Ara-Lynda Norwiya K. (Principal Author)
Sanaani, Wallie Jr. M.
Locker No.32; NurBio Lab E
1st Semester, SY 2019-20
Nursing Biochemistry
Laboratory Report

RATIONALE
This experiment was conducted to deduce the function of Enzymes through the following
tests: Test for Catalase and Test for Specificity. The objective for conducting this experiment is to
elucidate these tests derived from the experiment and some properties of lipids used in the
experiment, their definition and the basic concepts of the Enzymes and their properties.

Enzymes are natural biological catalysts that speed up the reactions occurring in the living
organisms, both animals and plants. The use of a specific enzyme makes a typical metabolic
reaction a million times faster. Enzymes are being designed specifically for a certain chemical
reaction in the system. This is through DNA, in the cell’s nucleus, which encodes instructions for
the cells to synthesized specific enzymes. An enzymatic activity consists of substrate, active site
and enzymes. Enzymes act on substrates. The specific site where enzymatic activity takes place is
the active site. The substance formed by the reaction is called product. The salivary amylase,
produced by salivary glands, is a type of hydrolase enzyme (amylase). Hydrolases catalyze
hydrolysis reactions where there is an addition of a water molecule to a bond resulting in bond
breakage. Amylase breaks down starch and turns in into maltose, a disaccharide. These reactions
have an important role in digestive process.

Test for Catalase and Test for Specificity of Enzyme Action were the two tests or
experiment that was conducted in the class in order for us to determine the result of the following
tests: Biuret, Catalase and Iodine or the Specificity of Enzyme Action tests.

According to the internet based knowledge, “The biuret test, also known as
Piotrowski's test, is a chemical test used for detecting the presence of peptide bonds. The test is
named so because it also gives a positive reaction to the peptide-like bonds in the biuret molecule.
In this assay, the copper(II) binds with nitrogens present in the peptides of proteins.” -Wikipedia,
2019. While Catalase acts as the catalyzing enzyme in the decomposition of hydrogen peroxide.
Nearly all living things possess catalase, including us. This enzyme, like many others, aids in the
decomposition of one substance into another. Catalase decomposes, or breaks down, hydrogen
peroxide into water and oxygen. While in the other hand, Iodine test is to detect the presence or
absence of starch in the solutions using iodine (I2). Iodine forms a blue to black complex with
starch, but does not react with glucose. If iodine is added to a glucose solution, the only color seen
is the red or yellow color of the iodine. Therefore, the faster the blue color of starch is lost, the
faster the enzyme amylase is working. If the amylase is inactivated, it can no longer hydrolyze
starch, so the blue color of the starch-iodine complex will persist.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

DATA INTERPRETATION
“The biuret test, also known as Piotrowski's test, is a chemical test used for detecting the presence
of peptide bonds. The test is named so because it also gives a positive reaction to the peptide-like
bonds in the biuret molecule. In this assay, the copper(II) binds with nitrogens present in the
peptides of proteins.” -Wikipedia, 2019

1. Test for Catalase

Table No. 1

Test Reagents Added Result

Biuret 10% NaOH, 1% CuSO4 Violet Color Appearance

Catalase Activity (Potato Gas evolved, Solution remain

3% H2O2, 0.5% Benzidine
extract) in color

Documentation

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Nursing Biochemistry
Laboratory Report

Discussion

In the experiment, we were given the task to perform the Biuret test wherein the reagent
solution that were added are 10% NaOH and 1% CuSO4 that resulted to a Violet Color appearance
and Catalase Activity on Potato extract wherein we were given the task to observe the gas, and the
reagents that were added are 3% H2O2 and 0.5% Benzidine which also falls under the test for
Catalase that gave a result of gas being evolved and the solution color remains.

The biuret test is a chemical assay that detects the presence of proteins in a sample. The test relies
on a color change to confirm the presence of proteins. If proteins are found, the sample will turn
violet. The biuret test doesn't involve the chemical biuret, which is derived from urea. Biuret isn't
a protein, but it gives a positive result to the biuret test. The biuret test uses an alkaline mixture, or
reagent, composed of potassium hydroxide and copper sulfate. The normal color of biuret reagent
is blue. The reagent turns violet in the presence of peptide bonds -- the chemical bonds that hold
amino acids together. The proteins detected must have at least three amino acids, which means
that the protein must have at least two peptide bonds. The reagent’s copper ions, with a charge of
+2, are reduced to a charge of +1 in the presence of peptide bonds, causing the color change. The
techniques of absorption spectroscopy, which identify the electromagnetic frequencies a sample
will absorb, allow testers to quantify the concentration of protein in a sample.

The normal color of biuret reagent is blue. The reagent turns violet in the presence of
peptide bonds -- the chemical bonds that hold amino acids together. The reagent's copper ions,
with a charge of +2, are reduced to a charge of +1 in the presence of peptide bonds, causing
the color change. The violet color appearance reaction happens when Cu2+ typically placed into
solution as cupric sulfate is reduced to Cu1+ as it complexes with peptide bonds in proteins. When
it does, the solution turns a purple color – lighter if there is less protein and darker if there is more.

2. Test for Specificity of Enzyme Action

The use of Lugol's iodine reagent (IKI) is useful to distinguish starch and glycogen from
other polysaccharides. Lugol's iodine yields a blue-black color in the presence of starch. Glycogen
reacts with Lugol's reagent to give a brown-blue color. Other polysaccharides and
monosaccharides yield no color change; the test solution remains the characteristic brown-yellow
of the reagent. It is thought that starch and glycogen form helical coils. Iodine atoms can then fit
into the helices to form a starch-iodine or glycogen-iodine complex. Starch in the form of amylose
and amylopectin has less branches than glycogen. This means that the helices of starch are longer
than glycogen, therefore binding more iodine atoms. The result is that the color produced by a
starch-iodine complex is more intense than that obtained with a glycogen-iodine complex.
Solutions are brown or orange in strongly polar solvents, for example, ethanol and acetone.
When iodine is dissolved in carbon disulphide, carbon tetrachloride, or chloroform, it yields
purple-colored solutions. Iodine is slightly soluble in water and gives a yellow solution.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

Table No. 2

Test Tube 1- Starch Test Tube 2 – Glycogen

Time Interval, minutes
Color with Iodine Color with Iodine

5 Light Mahogany Red Light Mahogany Red

10 Light Mahogany Red Light Mahogany Red

15 Dark Mahogany Red Light Mahogany Red

20 Dark Mahogany Red Dark Mahogany Red

25 Darker Mahogany Red Darker Mahogany Red

30 Darker Mahogany Red Darker Mahogany Red

35 Dark Mahogany Red Dark Mahogany Red

40 Dark Mahogany Red Dark Mahogany Red

45 Dark Mahogany Red Light Mahogany Red

50 Dark Mahogany Red Light Mahogany Red

55 Dark Mahogany Red Dark Mahogany Red

60 Darker Mahogany Red Darker Mahogany Red

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

Documentation

Discussion

In this experiment, we were given the task to observe the difference of coloration
of Iodine test between Starch and Glycogen for an hour. And we were given the result
which is shown in the photo provided above.

Iodine (atomic number 53, symbol I) is a chemical element with low toxicity, which
dissolves easily in chloroform, hexane, and other organic solvents due to its lack of
polarity. The color of iodine solutions depends on the solvent and its polarity. Solutions
are violet in color in hexane and other non-polar solvents and dark crimson in moderately
polar ones. Solutions are brown or orange in strongly polar solvents, for example, ethanol
and acetone. When iodine is dissolved in carbon disulphide, carbon tetrachloride, or
chloroform, it yields purple-colored solutions. Iodine is slightly soluble in water and gives
a yellow solution.

This element is shiny, blue-black, nonmetallic solid, volatizing into a violet-blue gas at
room temperature. It has an irritating odor as well. Iodine is less reactive compared to other
halogens and forms compounds with different elements. Iodine also exhibits some
properties characteristic of metals. It binds to starch, coloring it dark blue.

Iodine has multiple applications as well. It is used for water treatment, as a disinfectant,
and as a radiocontrast agent. There are special applications of inorganic iodides. Titanium,
zirconium, and hafnium are purified by using the van Arkel Process. Then, the main
component of photographic film is silver iodide, while cloud seeding consumes thousands
of kilograms of the compound a year. Some disorders of the thyroid gland are treated by
using iodine-131, which is a radioactive isotope of the element.

Iodine is the main component of thyroid hormones, which are essential for metabolism, the
nervous system, and growth. Iodine shortages are experienced by persons who eat no or
little bread. The thyroid gland’s function slows down, and it swells up. The intake of large
quantities is dangerous and can affect all organs in the body, causing loss of weight and
disturbed heartbeats.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

ANSWERS TO QUESTIONS

1. What is the chemical nature of enzyme?

All known enzymes are proteins. They are high molecular weight compounds made up
principally of chains of amino acids linked together by peptide bonds. See Figure 1.

Enzymes can be denatured and precipitated with salts, solvents and other reagents. They
have molecular weights ranging from 10,000 to 2,000,000.

Many enzymes require the presence of other compounds - cofactors - before their catalytic
activity can be exerted. This entire active complex is referred to as the holoenzyme; i.e.,
apoenzyme (protein portion) plus the cofactor (coenzyme, prosthetic group or metal-ion-activator)
is called the holoenzyme.

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Nursing Biochemistry
Laboratory Report

Apoenzyme + Cofactor = Holoenzyme

According to Holum, the cofactor may be:
1. A coenzyme - a non-protein organic substance which is dialyzable, thermostable and
loosely attached to the protein part.
2. A prosthetic group - an organic substance which is dialyzable and thermostable
which is firmly attached to the protein or apoenzyme portion.
3. A metal-ion-activator - these include K+, Fe++, Fe+++, Cu++, Co++, Zn++, Mn++, Mg++,
Ca++, and Mo+++

2. What type of enzyme is catalase?

Catalase is an antioxidant enzyme present in all aerobic organisms. It is known to catalyze

H2O2 into water and oxygen in an energy-efficient manner in the cells exposed to
environmental stress. Catalase is located in all major sites of H2O2 production in the cellular
environment (such as peroxisomes, mitochondria, cytosol and chloroplast) of higher plants.
Multiple molecular forms of catalase isozymes indicate its versatile role within the plant
system. The modulation of H2O2 by the catalase isozymes within specific cells or organelles at
specific time and developmental phases directly or indirectly interferes with signal
transduction in plants. Catalase isozymes CAT-1, CAT-2, CAT-3 have been encoded by
structural genes Cat1, Cat2 and Cat3, respectively. The expression of cat gene shows time,
species and stress specificity. Catalase deficiency in plants develops anomalies such
as chlorosis and head sterility and sensitivity to normal photorespiratory conditions. The
molecular phylogeny of plant catalase proteins also reveals the structure and functional links
among a wide range of plant species. This chapter compiles the latest information on catalase
structure, localization, biochemistry, genes and function in plants.

Catalases are antioxidant enzymes that catalyze the conversion of hydrogen peroxide to
water and molecular oxygen. According to the structure and sequence, catalases can be divided
into three classes (Fig. 11.2): monofunctional catalase or typical catalase, catalase-peroxidase, and
pseudocatalase or Mn-catalasee (Zhang et al., 2010). Currently, there are at least eight strains that
can produce catalases (Zhang et al., 2010): Penicillum variable, A. niger, S.
cerevisiae, Staphylococcus, Micrococcus lysodeiktious, Thermoascus
aurantiacus, Bacillus subtilis, and Rhizobium radiobacte. Catalases are used in several industrial
applications such as food or textile processing to remove hydrogen peroxide that is used for
sterilization or bleaching.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

3. What is the specificity of salivary amylase?

The specificity of an enzyme refers to the types of chemical bonds with which the enzyme
reacts. Each enzyme has a particular range of what it can and cannot react with as well as a range
of conditions under which it will either be active or inactive. Most enzymes have a very specific
target and will only react when a particular chemical or chemical bond is present. Others are highly
reactive and will produce chemical reactions with almost any substance.
The specificity of salivary amylase is highly targeted to react with glucose found in molecules of
various starches. When salivary amylase come in.

4. What mechanism is involved in the hydrolysis of starch by salivary amylase?

Hydrolysis of starch or oligosaccharides by mammalian amylases, in general, results in maltose
as the leaving group. The active site of these amylases harbors three aromatic residues Trp59,
Tyr62, and Tyr151, which provide stacking interactions to the bound glucose moieties. We
hypothesized that Tyr151, located at the S2' subsite, may influence the size of the leaving group.
Therefore, using a baculovirus expression system, we generated a mutant Y151M in which the
tyrosine at position 151 of human salivary amylase is replaced by a methionine. The specific
activity, K(m), rate of hydrolysis, and the product distribution for Y151M were distinctly different
from those of the wild-type enzyme using starch and oligosaccharides as substrates. The mutant
enzyme Y151M consistently produced glucose as the minimal leaving group and exhibited a
twofold increase in K(m). These results suggest that the stacking interaction at subsite S2' in the
wild type plays a role in hydrolysis.

5. What are the degradation products of the enzymatic hydrolysis of starch?

It greatly depends on the type of enzymes used for hydrolysis.

The term ‘hydrolysis’, bigger molecules get broken down into smaller ones by reacting with water.
Enzymes play a great role in breaking down the molecules.

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Nursing Biochemistry
Laboratory Report

Whenever starch (polysaccharides) molecules undergo hydrolysis, it forms either

monosaccharides, disaccharides or trisaccharides.

The end products depends on the strength of enzymes used and the common enzymes are,

 α-Amylase, which produces the disaccharide maltose and the trisaccharide maltotriose
 β-Amylase, which produces the disaccharide maltose
 γ-Amylase, which produces glucose

Enzymes are not some kind of hazardous laboratory biochemical substance. α-Amylase is an
enzyme present in human saliva and β-Amylase in potatoes and other starchy vegetables. Starch
hydrolysis is, in fact, happening in human body every time we consume carbohydrates.

6. Account for the addition of NaCl and phosphate buffer.

NaCl and phosphate buffer are used to control the pH of the media to ensure the right pH for
the desired action since the enzymes are very pH sensitive.

CONCLUSION
Taking everything into account and everything already being considered, overall the main
objective is to determine the tests that were mentioned which includes the biuret test that gives a
black to violet for a positive reaction or result and catalase activity on potato extract giving blue
to green coloration as positive result or reaction and also the gas that evolved. For the Test of
Specificity of Enzyme Action, we were able to provide results for the starch and glycogen test for
iodine. Iodine (atomic number 53, symbol I) is a chemical element with low toxicity, which
dissolves easily in chloroform, hexane, and other organic solvents due to its lack of polarity.
Solutions are brown or orange in strongly polar solvents, for example, ethanol and acetone. When
iodine is dissolved in carbon disulphide, carbon tetrachloride, or chloroform, it yields purple-
colored solutions. Iodine is slightly soluble in water and gives a yellow solution.

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

REFERENCES
Elements Database. (2015) Retrieved from:
http://www.elementsdatabase.com/Iodine-I-53-element/

Unknown. (2012) Iodine Test for Starch and Glycogen. Retrieved from:
http://generalchemistrylab.blogspot.com/2011/12/iodine-test-for-starch-and-glycogen.html

Catalase and Hydrogen Peroxide Experiment. (2006) Retrieved from:

https://www.education.com/science-fair/article/activator/

Worthington Biochemical Corporation. (2019) Retrieved from:

http://www.worthington-biochem.com/introBiochem/chemNature.html

Iti, S. & Parvaiz, A. (2014) Catalase. Retrieved from:

https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/catalase

Ellicia, M., John, N., Nicanor, O., Maria, P. (2010) Enzymatic Activity of Salivary Amylase.
Retrieved from:
https://www.scribd.com/doc/49018719/Enzymatic-Activity-of-Salivary-Amylase

Specificity of Salivary Amylase. (2019) Retrieved from:

https://study.com/academy/answer/what-is-the-specificity-of-salivary-amylase.html

Vignesh, G. (2015) Products of Starch Hydrolysis. Retrieved from:

https://www.quora.com/What-products-are-made-by-starch-hydrolysis

Eric, B. (2019) What Does a Biuret Test Mean in Biology. Retrieved from:
https://education.seattlepi.com/biuret-test-mean-biology-4659.html

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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University