Experiment 1

Protein Quantification by using absorbance,

Bradford and Lowry methods
Part 1: Absorbance Assay
PRINCIPLE
Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm.
The peak at 280 nm is mainly because of the amino acids with aromatic rings.

EQUIPMENT

Liquid handling supplies, spectrophotometer, UV lamp and Quartz cuvette.

PROCEDURE

Carry out steps 1-4 (280 nm only) for a very rough estimate. Carry out all steps if
nucleic acid contamination is likely.

1.
2.
3.
4.
5.
6.
7.

Warm up the UV lamp (about 15 min.)

Adjust wavelength to 280 nm
Calibrate to zero absorbance with buffer solution only
Measure absorbance of the protein solution
Adjust wavelength to 260 nm
Calibrate to zero absorbance with buffer solution only
Measure absorbance of the protein solution

CALCULATIONS
Unknown proteins or protein mixtures. ( Path length for most spectrometers is 1 cm)
Concentration (mg/ml) = Absorbance at 280 nm divided by path length (cm.)
Pure protein of known absorbance coefficient. (Concentration is in mg/ml, %, or molarity
depending on which type coefficient is used)

Concentration = Absorbance at 280 nm divided by absorbance coefficient

Unknowns with possible nucleic acid contamination.
Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)

NOTES:

1. This method is simple, and requires an extremely small sample volume

2. However, the protein sample must be pure and does not contain any non-protein

components

with the same absorption spectrum, such as the contamination of nucleic acids.

3. This method is quickest, but error-prone.

Part 2: Bradford Method

PRINCIPLE
The assay is based on the observation that the absorbance maximum for an acidic
solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to
protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the
dye, causing a visible color change. The assay is useful since the extinction coefficient of a
dye-albumin complex solution is constant over a 10-fold concentration range.

EQUIPMENT
In addition to standard liquid handling supplies a visible light spectrophotometer is needed,
with maximum transmission in the region of 595 nm, on the border of the visible spectrum
(no special lamp or filter usually needed). Glass or polystyrene (cheap) cuvettes may be
used, however the color reagent stains both. Disposable cuvettes are recommended.

REAGENTS
1. Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95%
ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the dye has
completely dissolved, and filter through Whatman #1 paper just before use.

2.

(Optional) 1 M NaOH (to be used if samples are not readily soluble in the color reagent).
color. Filtration may have to be repeated to rid the
The Bradford reagent should be a light brown in
reagent of blue components. The Bio-Rad

concentrate is expensive, but the lots of dye used

have

apparently been screened for maximum effectiveness. "Homemade" reagent works quite
well but is usually not as sensitive as the Bio-Rad product.

PROCEDURE
Assay

1. Warm up the spectrophotometer before use.

2. Dilute unknownsgproteinifnecessin arytleasttoobtainoneassaybetween
5 and 100 tube containinglsample 100

3. If desirred, add an equal volume of 1 M NaOH to each sample and vortex (see
Comments below). Add NaOH to standards as well if this option is used.

4. Prepare standards containing a range of 5 to 10.

5. Add 5 ml dye reagent and incubate 5 min.
6. Measure the absorbance at 595 nm.

CALCULATIONS
Prepare a standard curve of absorbance versus micrograms protein and determine
amounts from the curve. Determine concentrations of original samples from the amount
protein, volume/sample, and dilution factor, if any.

NOTES:
1. One advantage of this method is its compatibility with reducing agents used to
stabilize proteins in solution, which are not compatible with Lowry assay and in
some extent with the BCA assay.
2. The limitation of Bradford assay is the incompatibility with low concentration of
detergents, which are routinely used to solubilize membrane proteins. However, the
detergent substances can be removed by gel filtration, dialysis, or precipitation of
the proteins with calcium phosphate.
3. A further advantage is the possibility to measure high molecular weight (MW)
proteins since the dye does not bind to peptides with low MW. However, very low
level of non-ionic detergent, such as Triton X-100 at a final concentration of 0.008%
(v/v), may improve sensitivity and variability of the Bradford assay.

Part 3: Hartree-Lowry Assay

PRINCIPLE
Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in
which it is reduced to a monovalent ion. Monovalent copper ion and the radical groups of
tyrosine, tryptophan, and cysteine react with Folin reagent to produce an unstable product
that becomes reduced to molybdenum/tungsten blue.

EQUIPMENT
In addition to standard liquid handling supplies a spectrophotometer with infrared lamp
and filter is required. Glass or polystyrene (cheap) cuvettes may be used.

REAGENTS
1. Reagent A consists of 2 gm sodium potassium tartrate x 4 H20, 100 gm sodium
carbonate, 500 ml 1N NaOH, H20 to one liter (that is, 7mM Na-K tartrate, 0.81M
sodium carbonate, 0.5N NaOH final concentration). Keeps 2 to 3 months.
2. Reagent B consists of 2 gm sodium potassium tartrate x 4 H20, 1 gm copper sulfate
(CuSO4 x 5H20), 90 ml H20, 10 ml 1N NaOH (final concentrations 70 mM Na-K
tartrate, 40 mM copper sulfate). Keeps 2 to 3 months.
3. Reagent C consists of 1 vol Folin-Ciocalteau reagent diluted with 15 vols water.

PROCEDURE
1.

Prepare a series of dilutions of 0.3 mg/ml bovine serum albumin in the same buffer containing the
unknowns, to give concentrations of 30 to 150 micrograms/ml (0.03 to 0.15 mg/ml).

2. Add 1.0 ml each dilution of standard, protein-containing unknown, or buffer

(for the reference) to 0.90 ml reagent A in separate test tubes and mix.

3. Incubate the tubes 10 min in a 50 degrees C bath, then cool to room temperature.
4. Add 0.1 ml reagent B to each tube, mix, incubate 10 min at room temperature.
5. Rapidly add 3 ml reagent C to each tube, mix, incubate 10 min in the 50 degree bath,
and cool to room temperature. Final assay volume is 5 ml.

6. Measure absorbance at 650 nm in 1 cm cuvettes.

CALCULATIONS
Prepare a standard curve of absorbance versus micrograms protein (or vice versa),
and determine amounts from the curve. Determine concentrations of original samples
from the amount protein, volume/sample, and dilution factor, if any.

NOTES:
1. The advantages of this assay are its sensitivity, and most importantly, accuracy.
However, it requires more time than other assays and many compounds commonly
used in buffers for protein preparation (such as, detergents, carbohydrates, glycerol,
Tricine, EDTA, Tris) interfere with Lowry assay and form precipitates .

2. Nonetheless, the effect of these substances can be reduced by diluting out the
sample but only if the protein concentration is sufficiently high [8]. Moreover, it has
been shown that the time to perform this assay can be reduced through raising
temperatures or using a microwave oven .

Experiment 2
Assay of - Glucosidase
( - Glucosidase Catalyzed Hydrolysis of PNPG)

THEORY

eta - Glucosidase is a glucosidase enzyme that acts upon 1 ->4 bonds linking two glucose or
glucose- substituted molecules and hydrolyzes of glucosides. It catalyses the hydrolysis of terminal non
reducing residues in beta-D- glucosides with release of glucose. glucosidase activity can be
conveniently assayed by monitoring the formation of a chromogenic product p-nitrophenol , which is
formed by hydrolysis of p-nitrophenol - D glucoside(PNPG). The amount of PNP formed can be
measured by determing the absorbance at 410nm. Amount of PNP produced is proportional to the

amount
of - glucosidase and at the time of the reaction. The reaction is stopped by adding Na2CO3

which shifts the pH of reaction mixture to alkaline( pH = 11.0). At this pH most of the PNP is
converted to the yellow colored anionic form and
- glucosidase is inactivated.
- Glucosidase

p-Nitrophenyl--D-glucoside (PNPG)

p -Nitrophenol (PNP) _D-Glucose

PROCEDURE

1- Preparation of reagents:

(1) Citrate buffer 50mM, pH 4.8: Prepared by adding 23.0 ml of 50 mM Citric acid
(2)
(3)
(4)
(5)

solution and 27.0 ml of 50mM sodium citrate solution, diluted to a total of 100 ml.
PNPG solution: 1mM solution prepared by dissolving 30mg PNPG in 100 ml of
50mM citrate buffer.
PNP solution 1mM: prepared by dissolving 13.9 mg of PNP in 100ml of citrate buffer.
Sodium carbonate solution(1N) : by dissolving 10.6 g Na 2CO3 in 100 ml Distilled water.
Glucose 100mM: Dissolve 1.8g in 100 ml of citrate buffer.

2- Equipment/ Instruments required

(1) Spectrophotometer
(2) Water bath
(3) Vortex mixer
3- Preparation of standard curve:

(1) Dilute the PNP solution to a final volume of 0.2 ml with citrate buffer to give a
range of PNP concentrations( 0.1-1mM).
(2) To 0.2 ml of diluted PNP solution, add 1.8 ml of citrate buffer.
(3) Add 1ml of 1N Na2CO3 solution to each test tube and measure the OD at
400nm against the reagent blank.
(4) Plot OD at 400-nm vs concentration of PNP
4-Enzyme assay:

(a) To 0.2ml of suitably diluted enzyme, add 1.8 ml of PNPG solution, preincubated
at 50C for 10 minutes.
(b)Incubate the mixture for 10 minutes and stop the reaction by adding 1ml of
sodium carbonate solution.
(c)Measure the absorbance at 400nm.
(d)Calculate the enzyme activity as:

CALCULATIONS

IU/ml =

Experiment 3
Assay of Invertase Activity
PRINCIPLE
Invertase or - fructofuranosidase catalyzes the hydrolysis of sucrose to yield invert
sugar, resulting in a mixture of glcose and fructose. Inverted sugar is sweeter than
sucrose and has a much lesser tendency to crystallize. Enzyme activity is assayed
by estimating the amount of glucose( reducing sugar) produced by DNS method.
Reducing sugars have the property to reduce many reagents( e.g. 3,5 dinitro
salicylic acid or DNS) in alkaline solution. DNS is reduced to 3, amino , 5 nitro
salicylic acid which is orange- red in color and strongly absorbs light at 540 nm.

REAGENTS:
1.
2.
3.
4.

Acetate buffer(0.05M , pH 5.0)

0.1 M sucrose in acetate buffer
0.5 N NaOH
Glucose (1mg/ml)

PROCEDURE:
Standard Curve:

1. Prepare various dilutions (0.2 to 1 mg/ml) of standard glucose to make the

volume upto 1 ml.
2. Add 1ml of 0.05M acetate buffer to each tube
3. Add 3ml of DNS reagent and put the tube in boiling water batch for 5 min.

4.

Add 15 ml of distilled water to all the tubes and estimate OD at 540 nm against blank.

Enzyme Assay:

1. Take 1 ml of suitably diluted enzyme preparation and add 1ml of 0.1M sucrose solution.
2. Incubate the mixture at room temperature for 5 min.
3. After incubation to 1 ml of above mixture add 1ml of 0.05 N NaOH to stop the reaction.
4. Add 3ml of DNS reagent and keep the tubes in boiling water for 5 min
5. Add 15 ml of distilled water to each tube and note the absorbance at 540nm.

CALCULATIONS
Invertase(U/ml) =

Experiment 4
Assay of Yeast Alcohol Dehydrogenase Activity
PRINCIPLE:
Alcohol dehydrogenase catalyses the reaction:
RCH2OH + NAD+

RCHO + NADH + H+

Their common reaction in yeast cells is reduction of acetaldehyde to ethanol. The assay is
done by measuring reaction velocity by the method of Vallee & Hoch in which the rate of
increase in absorbance at 340 nm resulting from the reduction of NAD + is measured. One
unit reduces one micromole of NAD+ per minute at 25oC.
Enzyme activity is calculated by the following formula:

REAGENTS:
1
2
3
4
5

0.03M Sodium pyrophosphate buffer (pH 8.8)

2 M ethanol prepared by diluting 12.12 ml of 95% ethanol to 100 ml of distilled water
0.1 M Phosphate buffer, pH 7.5
0.01 M Phosphate buffer, pH 7.5
0.025M NAD+

PROCEDURE:
1. The various constituents are pipetted into test tubes as follows:
For Blank (ml)

For Test (ml)

Phosphate buffer (pH 8.8)

1.9

1.8

NAD+

1.0

1.0

Ethanol

0.1

0.1

2. Pour the contents into a 3 ml cuvette and set the wavelength at 340 nm.
3. The spectrophotometer should be set in the time drive mode.
4. Measurement of enzyme activity is started as soon as 0.1 ml of suitably diluted enzyme
sample is added to the cuvette in the case of test sample.

5. The initial rate is measured for 2 minutes of reaction and the enzyme activity calculated as
stated above.

NOTES:
1. Alcohol Dehydrogenase is unstable in solution and should be assayed immediately

following preparation of solutions

Experiment 5
Kinetic Analysis of Enzyme Catalysed Reaction (Determination of

Km and Vm)
PRINCIPLE:
For an enzyme exhibiting Michaelis-Menten kinetics, velocity may be represented as:

1=

Or,
1=

+ = 1 +

The kinetic parameters Km and Vmax can be determined by plotting a graph between 1/V vs.
1/S referred to as the Lineweaver-Burk Plot.

However, the above plot and others (Eadie-Hofstee) dont allow accurate estimation of the
kinetic constants.
Eisenthal and Cornish-Bowden , suggested a modification of the Michaelis-Menten equation,
which can also be expressed as:

=1+

Set up axes, Km and V, corresponding to the familiar x and y axes respectively. For each
observation (S,V), mark off the points K m = -S on the Km axis and Vmax = v on the Vmax axis and
draw a line through the two points extending it to the first quadrant. When this is done for all
observations, the lines intersect at a common point whose coordinate (K m, Vmax) provide the
values of Km and Vmax that satisfy the Michaelis-Menten equation exactly for every observation.

REAGENTS:
1. Citrate buffer (50 mM, pH 4.8) prepared by adjusting pH of 50 mM citric acid with 1 N
NaOH (dissolve 9.6 gm citric acid in water and adjust pH with NaOH solution to make
up volume to 1 l)
2. PNPG (2.5 mM) stock solution in citrate buffer (50 mM, pH 4.8) (7.53 g in 10 ml)

3. Na2CO3 (1N) solution in distilled water (10.6 g in 100 ml)

PROCEDURE:
1. Take 1.8 ml of PNPG solution containing five different substrate concentrations (0.52.5 mM) in 5 different test tubes
2. Prepare two sets to one add 0.2 ml suitable diluted enzyme and to the other add 0.2
ml citrate buffer
3. Incubate the tubes at 50oC in water bath for 15 minutes

4. Stop the reaction by adding 1 ml Na2CO3 solution and measure A410 nm.
5. Determine initial velocity (moles/min) using the standard plot at the initial substrate
concentration taken.

6. Prepare the Lineweaver-Burk plot (1/V vs 1/S) and determine the values of K m and
Vmzx for the reaction.
7. Next, plot the Cornish-Bowden plot each of the data points and determine the point of
intersection to calculate the true values of Km and Vmax

Experiment 6
Kinetic Characterization of the enzyme and study of
substrate inhibition
OBJECTIVES:
1. To find the reaction kinetics for alcohol dehydrogenase and isocitrate dehydrogenase
2. To study the nature of the inhibition of the enzyme ( -glucosidase) and alcohol

dehydrogenase by provided inhibitors and to determine inhibition constant (Ki). Study

the change in rate of reaction of an allosteric enzyme in the presence of inhibitors.

PRINCIPLE:
For an enzyme showing the typical Michaelis-Menten kinetics:

1=

Or,
1

So the kinetic parameters, Km and Vm can be determined by plotting a graph of between 1/V
vs 1/S. this plot is called Lineweaver-Burk plot. The intercept on the Y-axis gives 1/V m and
the slope equals Km/Vm
In view of difficulty in obtaining the reliable estimates of K m and Vm. Eisenthal and CornishBowden suggested a new approach.
Therefore at a constant S and V, a hypothetical plot of V m vs Km is linear. When Km = 0, Vm = v
and at at Vm = 0, Km = -S. Each data point of the reaction, i.e., v i and Si will yield a straight line
and intersection of these lines will give a reliable estimate of the true K m and Vm.
The three main mechanisms for the inhibition of the enzyme-catalyzed reactions can be

kinetically expressed as:

1. Competitive inhibition:
1/V = 1/Vm + Km/Vm (1+1/Ki).1/S
Vm is unaffected but the apparent Km is increased by a factor (1+1/Ki)
2. Non-competitive inhibition:
1/V = 1/Vm (1+1/Ki) + Km/Vm(1+1/Ki).1/S
Km remains unaffected but Vm is decreased by a factor 1/(1+1/Ki)
3. Mixed Inhibition:

1/V = 1/Vm(1+1/Ki) + Km/Vm.1/S

Here both Km and Vm are affected by inhibitor.

REAGENTS:
1 Citrate buffer (50 mM, pH 4.8), prepared by adjusting pH of 50 mM citric acid with 1N

2
3
4
5

NaOH (dissolve 9.6 g citric acid in water and adjust pH with NaOH solution to make
up volume to 1 l)
PNPG (2.5 mM) stock solution in citrate buffer (50 mM, pH 4.8) (37.65 mg in 50 ml)
Na2CO3 (1N) solution in distilled water (10.6 g in 100ml)
Glucose 100 mM (1.8 g in 100 ml)
-glucosidase (2.5 mg in 5 ml)

PROCEDURE:
1. Take 1.8 ml of PNPG solution containing five different substrate concentrations (0.52.5 mM) in 5 different test tubes
2. Prepare two sets to one add 0.2 ml suitable diluted enzyme and to the other add 0.2
ml citrate buffer

3. Incubate the tubes at 50oC in water bath for 15 minutes

4. Stop the reaction by adding 1 ml Na2CO3 solution and measure A410 nm.
5. Determine initial velocity (moles/min) using the standard plot at the initial substrate
concentration taken.
6. Prepare the Lineweaver-Burk plot (1/V vs 1/S) and determine the values of K m and
Vmzx for the reaction and calculate Ki using the above equation.

Experiment 7
Investigating the effect of pH and temperature on enzyme activity
PRINCIPLE:
In this lab, we will explore some of the properties of enzymes. Enzymes are sensitive to
changes in temperature and pH which alter their shapes and can even destroy catalytic
ability (denaturing). Enzymes have evolved to work most efficiently at the temperature and
pH found in the part of the organism where they are needed. Many enzymes in the human
body function most efficiently at 37oC and at a pH of 7.4
The enzyme you will investigate is called catalase. Catalase is found in tissues of many
organisms (both plants and animals) because it plays a very important role in protecting cells. Its
purpose is to destroy toxic substances which may be introduced into cells. Also, some cells use
catalase to destroy cellular debris or worn out organalles. Hydrogen peroxide is a normal byproduct of cellular metabolism but it is also toxic to cells. under normal conditions organisms
produce the enzyme catalase that quickly changes hydrogen peroxide into two harmless
substances, oxygen and water. However, the function of the enzyme is affected by changes in
the environment. Catalase works to breakdown hydrogen peroxide by the following reaction:

2H2O2 -----> 2H2O + O2

MATERIALS:
Catalase (to be prepared by you), 1% hydrogen peroxide, forceps, filter paper discs, water
baths, two large test tubes, 9 small test tubes

IMPORTANT NOTE:
The assay system used in the lab consists of a filter paper disc which is coated with the
enzyme and then dropped into a test tube of substrate (hydrogen peroxide). As the catalyst
breaks down the hydrogen peroxide into water and oxygen gas, the bubbles of oxygen
collect underneath the filter and make it rise to the surface of the hydrogen peroxide. The
time it takes for the filter to rise will be ur indication of the rate of enzyme activity.
Rate of enzyme activity = distance (depth of hydrogen peroxide in mm)/time (in sec)
We will assume that each filter is coated with the same amount of catalase.
Catalase can be prepared easily by the student as follows:
50g of peeled potato was mixed with 50 ml cold distilled water and crushed ice and
homogenized in a blender for 30 seconds. This extract was filtered through cheesecloth and
cold distilled water was added to a total volume of 100 ml.
Part A: Effect of pH

1. Obtain 1 large test tube of 20 ml H2O2. Measure and record the depth of H2O2.
2. Label 5 small test tubes as pH3, pH5, pH7, pH9, pH11 and as
follows: pH3: 2 ml catalase + 2ml pH 3 buffer
pH5: 2 ml catalase + 2ml pH 5 buffer

pH7: 2 ml catalase + 2ml pH 7 buffer

pH9: 2 ml catalase + 2ml pH 9 buffer
pH11: 2 ml catalase + 2ml pH 11 buffer

Control: just dip your filter paper into pure catalase. No pH buffers will be added.

3. Dip a filter into the catalase at pH3, drain on a paper towel and place at the bottom of H 2O2.
4. Time how long it takes the filter to rise to the top.
5. Remove the filter and repeat the procedure for each of the pH and just dip a filter
paper into pure catalase for the control.
6. Record your results
Part B: Effect of Temperature

1. Set up the following water baths: ice bath (0 oC), body temperature (37oC), boiling
2.
3.
4.
5.
6.
7.
8.

water bath (100oC). For room temperature, just set catalase on the tabletop.
Obtain 1 large test tube of 20 ml H2O2. Measure and record the depth of H2O2.
Label 4 small test tubes as follows: 0oC, 22oC, 37oC, 100oC. Add 5 ml of catalase to
each test tube.
Place each test tube in correct water bath for 5 minutes.
Dip a filter into the enzyme from ice bath, drain on a paper towel and drop into 1% H 2O2.
Time how long it takes for filter to rise.
Repeat for each temperature.
Record your results.

ANALYSI
S:

Include two graphs.

1. pH vs average rate
2. Temperature vs average rate
Questions to consider in your analysis:

1. How does temperature affect the activity of catalase? Explain your observations by discussing
the effect of temperature on protein structure. Discuss both high and low temperature effects.

2. How does pH affect the activity of catalase? Consider both high and low pH and explain
your observations by discussing the effect of pH on protein structure.

3. Ectothermic organisms have body temperatures that vary with the temperature of their
surroundings (like reptiles). Discuss the effect this variation might have on functioning of
enzymes in these organisms.

Experiment 8
Enzyme entrapment in calcium alginate beads
PRINCIPLE:
Immobilization of enzyme by gel entrapment involves the entrapment of the biocatalyst
within a polymeric network. The biocatalyst after mixing with the aqueous polymeric solution
is forced through a fine orifice (syringe) into a salt solution that insolubilizes the mixture
through ion exchange. The shape and size of beads can be controlled by choosing the
orifice diameter and the distance of the nozzle from the liquid surface.
Gel entrapment usually does not result in any adverse modification of the enzyme
conformation and can provide high yield of immobilization.

REAGENTS:
1
2
3
4
5

Acetate buffer (0.05 M, pH 5)

Sodium alginate 4% w/v in acetate buffer
Enzyme source (Bakers yeast permeabilized cells as source of invertase)
0.1 M sucrose in acetate buffer
0.5 N NaOH

PROCEDURE:
1.
2.
3.
4.
5.

Mix the cell suspension to the sodium alginate solution (equal volumes) to bring the final
concentration of sodium alginate to 2% w/v.
Add this slurry dropwise into chilled calcium chloride solution under continuous stirring.
Cure the calcium alginate beads in calcium chloride solution for 30 minutes and wash
thoroughly with the buffer.
Suspend the gel beads in acetate buffer.
Assay for the invertase activity in the beads and compare the same with that in the original cell
suspension on the basis of equal weight of cell mass. Report the results in terms of yield of
immobilization, enzyme loading, bead diameter and packing density of the preparation.

Enzyme Assay

1.
2.
3.
4.
5.

Take 1 ml of suitably diluted enzyme preparation and add 1 ml of 0.1M sucrose solution.

Incubate the mixture at room temperature for 5 min.

After incubation to 1 ml of above mixture add 1 ml of 0.05N NaOH to stop the reaction.
Add 3 ml of DNS reagent and keep the tubes in boiling water bath for 5 min.
Add 15 ml of distilled water to each tube and note the absorbance at 540 nm.
(

min

Experiment 9

To hydrolyze protein-based stains in fabrics into soluble

amino acids.
PRINCIPLE:
In today's laundry detergents, enzymes such as proteases and amylases are some of the active
ingredients. In the U.S., about 50% of liquid detergents, 25% of powder detergents, and almost all
powdered bleach additives now contain enzymes to help break down stains that are otherwise hard to
remove with conventional surfactants alone. For example, amylase catalyzes the breakdown of starchbased stains to smaller segments that make up the larger starch molecule. Oligosaccharides and dextrins
released from the enzyme's hydrolytic action are soluble; thus, the stain is physically cut off from the
surface of the fabric piece by piece, with the enzyme acting as scissors. The action of proteases, as
implied by the name itself, is similar to that of amylase, except that a large protein molecule is hydrolyzed.
During the process of hydrolysis, the peptide bonds that hold various amino acids together to form a
protein molecule are broken down, releasing smaller polypeptides and individual amino acid units.
Generally, polymers made of less than approximately one hundred amino acid monomer units are called
polypeptides and larger ones are called proteins.

A small piece of fabric soiled by the dyed casein will be provided to each student. The purpose
of this experiment is to observe the hydrolytic action of bacterial protease in removing proteinbased stains. Because detergents, especially bath soaps, are generally formulated to degrade
mainly oil and grease, protein-based stains have traditionally been among the hardest to
remove. Proteins can act as strong natural bonding agents that make all sorts of dirt adhere
stubbornly to textile fibers. Anyone trying to wash away blood -stains can testify to this effect.
Other proteinaceous dirt includes perspiration, grass, and slime stains. This exercise
demonstrates that it takes protein to get out protein, as some television commercials claim.

MATERIALS REQUIRED:
1. Equipment
1
2
3
4
5

Flasks, 250 ml
Graduated cylinder
Household clothing iron or drying oven
Spectrophotometer
Balance

2. Reagents
1 Household detergent, (to be supplied by the student)
2 Bacterial protease
3 Dyed casein cloth

PROCEDURE:
1. Dissolve 1 g of household laundry detergent in 200 ml of hot (60C) water in a 250
ml flask. Colored detergent obviously interferes with the quantitative determination
of the rate of protein degradation by the colorimetric method; thus, it should not be
used if quantitative information is desired. Because the commercial detergent is not
completely soluble in water, filter the detergent solution before use.

2.

Similarly, mix 0.1 g of powder protease in 200 ml of hot water in a second flask. Because certain
protease is not water soluble, removal of the insoluble solids by filtration or centrifugation,
leaving behind only supernatant, may retain only very little protease activity.

3. Place a small piece of the dyed casein or gelatin cloth into each washing liquid.
4. Seal the top of each flask with paraffin or with a rubber stopper. Swirl both flasks
gently to simulate the agitating washing motion. A thermostated flask shaker may
be used for this purpose.
5. Periodically take out a small (5-10 ml) portion of the washing liquid and measure
the absorbance with a spectrophotometer until the changes in the color intensity
level off. The stain should be removed in approximately 20-30 minutes, but
sometimes it takes as long as a few hours for the complete removal of the color.
Because of the small particles of protein released during the wash, one may need
to filter the sample again with a 25mm syringe filtration unit fitted with a 10 ml
plastic syringe. This should be done immediately before measuring the absorbance.
6. After briefly rinsing the cloth under running tap water, dry it with a clothing iron.
7. Compare the effectiveness of the protease with your detergent. Also compare the
cloth washed with your detergent with the other students' to see how effective your
detergent is in cutting the stain.

8.

Simulate a "warm" (40C) and a "cold" (20C) wash cycle by repeating the above experiment at
appropriate temperatures. (Note that all the temperatures can be run simultaneously.)

Notes:
This experiment is for your better understanding, so once do think of ways to improve
this experiment as this exercise would help you build a better understanding.