EXPERIMENT 3
Enzymes
Pamela Estrada, Carlo Falconi, Shina Go, Joana Gozon, Monica Hamtig, Caitlin Hao.
Group 4 2F Medical Technology Organic Chemistry Laboratory
ABSTRACT
Enzymes are proteins that act as catalysts in speeding up the rate of reaction and help complex reactions occur
anywhere in life. Its performance mostly relies on several factors like pH, temperature, activators, cofactors, and
inhibitors. In this experiment, the invertase was extracted from Bakers yeast and used as the experimental enzyme.
Sucrose assays of different concentrations were prepared and tested on the particular enzyme using the
dinitrosalicylic colorimetric method. Buffer solutions with pH of 1:2, 2:3, 3:5, 4:7, 5:8, and 6:12; and water baths
with temperatures of 60C and 90C were also prepared and tested of their possible effects on the invertase activity
of the enzyme. The result of the experimental graph was a bell-shaped curve, which the group was able to
successfully obtain during the experiment.
INTRODUCTION
An enzyme is a protein molecule that is also
a biological catalyst with three characteristics. Its
first basic function is to increase the rate of a
reaction.
Cellular
reactions
often
occur
approximately a million times faster than they
would without the presence of an enzyme.
Secondly, most enzymes act specifically with only
one substrate to produce products. Lastly and
most importantly, enzymes are regulated from a
state of low activity to high activity and vice
versa. An enzyme usually found in plants that
catalyzes the breakdown of sucrose or table
sugar is called an invertase.
The performance of an enzyme mostly relies on
several factors like pH, temperature, activators,
cofactors, and inhibitors. Each enzyme has its
own range of pH wherein it will be most active.
The initial rates for many enzymatic reactions
exhibit bell-shaped curves, indicating the stability
of enzymes during the change of pH. [1]
Dinitrosalicylic acid ( D N S
or
3:5dinitrosalicylic acid) is a reagent used to
determine sugar content and is often used to
estimate the amount of sugar present in the
blood, in the cerebrospinal fluid, and in other
human bodily fluids. The amount of blood sugar
in the blood has metabolic implications and is
used to determine the presence of blood sugarrelated disorders such as hyperglycemia. One
good way to assess blood sugar level is through
the use of dinitrosalicylic acid. [2]
The objectives of this experiment are the
following:
(1) To extract invertase from Bakers yeast.
(2) To determine the effects of changes in pH
and temperature on reaction rates of an
enzyme-catalyzed reaction.
EXPERIMENTAL
A.
Compounds Tested
B.
Procedure
RESULTS AND DISCUSSION
1.
Solubility of Alcohols in Water
The table interprets that only benzyl alcohol was
insoluble in water, while ethanol, n-butyl alcohol,
sec-butyl alcohol and tertbutyl alcohol were all
soluble in water. The aphorism like dissolves
like implicates that the samples that are soluble
in water are polar. Some of the alcohols required
only 1 mL of water in order to be considered
soluble because of the factor of the number of
carbon atoms present in the sample. Branching
also affects the solubility of the compounds but it
is only true for compounds containing the same
number of carbon atoms present.
Table 1. Solubility of Alcohols in Water
Alcohol
Ethanol
n-butyl alcohol
sec-butyl
alcohol
Tert-butyl
alcohol
Benzyl alcohol
Amount of
water (in mL)
1mL
1.50mL
1mL
Solubility to
water
Soluble
Soluble
Soluble
1mL
soluble
3mL
insoluble
2.
Lucas Test
The Lucas test was used to differentiate 1, 2
and 3 alcohols. Turbidity and rate of reaction
were observed. Table 2 presents the results of
the Lucas test.
Table 2. Lucas Test
Substance
n-butyl alcohol
Sec-butyl alcohol
Tert-butyl alcohol
Observations
Colorless solution
Slightly turbid
Turbid solution
The table interprets that, n-butyl alcohol was
soluble in Lucas reagent while sec-butyl alcohol
and tert-butyl alcohol had a cloudy layer. Tertbutyl alcohol took the shortest time to form the
layer while sec-butyl alcohol took the longest
time. The reaction mechanism involved in the
Lucas test is based on SN1 reaction, which
depends on the formation of stable carbocations.
Reactivity of alcohols in SN1 reaction is 3 > 2>
1. The reaction rates in 3 alcohols formed the
second layer in less than a minute. 2 alcohols
required 5-10 minutes while 1 alcohols were
unreactive. The presence of ZnCl2, a good Lewis
acid, made the reaction mixture even more
acidic; thus, it enhanced the formation of
carbocations.
3.
Chromic Acid Test (Jones Oxidation)
This test detects the presence of primary and
secondary alcohols and aldehydes[9]. Primary
alcohols and aldehydes are oxidized into
carboxylic acid while secondary alcohols oxidize
in ketones but the three produces Cr3+ ions in the
process.
Tertiary
alcohols
do
not
react
immediately so they have negative results in this
tests. The formation of a blue to green colored
solution indicates a positive result. This is due to
the reduction of Cr6+ to Cr3+[10]. Acetone and
acetophenone serve as negative controls since
they do not oxidize when subjected to the
reagent. All the samples had positive results
except for tert-butyl alcohol since it is a tertiary
color. Even though it formed a dark-green
solution, it was not an immediate result.
Table 2. Chromic Acid Test (Jones Oxidation)
Substance
n-butyl alcohol
Tert-butyl alcohol
Acetaldehyde
Benzaldehyde
Acetone
Acetophenone
Isopropyl alcohol
Observations
Blue-green solution
Dark green solution
Blue-green solution
Blue-green solution
Dark green solution
Dark green solution
Blue-green solution
4.
2,4-Dinitrophenylhydrazone Test (2,4DNP Test)
Aldehydes and ketones show positive results in
this test by forming colored precipitates. A
negative result does not produce any precipitate
[11].Both produce precipitates, which are colored
yellow or orange. Non-conjugated carbonyl
compounds produce yellow precipitates while
conjugated ones form orange-red precipitates
[1]. Acetaldehyde, benzaldehyde, and acetone
produced yellow precipitates, which means that
they are not conjugated carbonyl compounds.
Acetophenone formed an orange precipitate
meaning that it is a conjugated carbonyl
compound.
Table 3. 2,4-DNP Test
Substance
Observations
Acetaldehyde
Benzaldehyde
Acetone
Acetophenone
Yellow precipitate
Yellow precipitate
Yellow precipitate
Orange precipitate
5.
Fehlings Test
This test verifies the presence of aldehydes by
producing brick-red precipitates due to curprous
oxide [12]. Most simple ketones do not react to
this test thereby not forming any precipitates [1].
Acetaldehyde and benzaldehyde formed brick-red
precipitates, which show that both compounds
are, in fact, aldehydes while acetone and
acetophenone did not form any precipitates
showing that they are not aldehydes.
Table 4. Fehlings Test
Substance
Acetaldehyde
Benzaldehyde
Acetone
Acetophenone
Observations
Brick-red precipitate
Brick-red precipitate
Blue solution
Blue solution
6.
Tollens Silver Mirror Test
This test is also known as the silver mirror test
and is used to distinguish between aldehydes and
ketones[13]. Aldehydes oxidize readily forming a
silver mirror or black precipitates, if the
glassware used is dirty[1]. Some compounds can
also form silver mirrors if those compounds can
be oxidized easily, giving false positive results[9].
Some phenols and aromatic amines are examples
of such. Ketones are not oxidized in this
experiment so they did not produce any
precipitate,
specifically
a
silver
mirror.
Acetaldehyde and benzaldehyde formed such so
they are oxidized into the corresponding
carboxylic acid[13], thereby being identified as
ketones.
Table 5. Tollens Silver Mirror Test
Substance
Acetaldehyde
Benzaldehyde
Acetone
Acetophenone
7.
Observations
Formation of silver mirror
Formation of silver mirror
No formation of silver
mirror; dark gray solution
No formation of silver
mirror; dark gray solution
Iodoform Test
Formation of haloform as a result of a chemical
reaction involving the methyl ketone halogenation
is a haloform reaction. Haloform reaction is used
for the production of iodoform (CHI 3) that is
yellow in color, and solid or crystalline in
appearance[9]. According to the 6th table,
acetone, acetophenone, and isopropyl alcohol
produced positive results of yellow crystals or
yellow precipitate. It can be observed that
acetaldehyde gave a yellow-brown solution yet
the positive result requires a yellow insoluble
precipitate. It can be concluded that aldehydes
(i.e.,
acetaldehyde,
n-butyraldehyde,
and
benzaldehyde) form negative results whereas
methyl ketones and 2o alcohols having adjacent
methyl group form positive results for iodoform
test.
Table 6. Iodoform Test
Substance
Acetaldehyde
n-butyraldehyde
Benzaldehyde
Acetone
Acetophenone
Isopropyl Alcohol
Observations
Yellow-brown solution
Orange-brown solution
Red-brown oil-like
precipitate
Pale yellow precipitate
Yellow crystalline
Clear solution with
yellow precipitate
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