Experiment 2 - Quantication of proteins by Bradford method

The Bradford method employs the binding of the dye, Coomassie Brilliant
Blue R-250, to proteins which causes a shift in the absorbance maximum of the dye
from 465 nm to 595 nm in acidic conditions. This method has only few known
interfering substances such as high concentrations of the detergents (e.g. triton x-
100 and Sodium Dodecyl Sulfate). The colour is developed when the dye (the
anionic form of the dye Coomassie Blue G-250) forms a strong, non-covalent
complex with proteins by electrostatic interactions with amino and carboxyl groups
via Van der Waals forces.
This dye reacts chiefly with arginine residues, which have a positively charged side
chain, and slight interactions have also been observed with basic residues (histidine
and lysine), and aromatic residues (tyrosine, tryptophan, and phenylalanine). In the
absence of protein, the dye reagent is a pale red, and upon binding to protein, a blue
colour is generated with an absorbance maximum at 595 nm.
Bradford assay is very popular because it is rapid (5 min) and involves a single
addition of the dye reagent to the sample. Therefore, this assay is quick and results
can be observed immediately.
Experimental hazards - Coomassie Brilliant Blue O-250
WARNING! CAUSES IRRITATION TO SKIN, EYES AND RESPIRATORY
TRACT. MAY BE HARMFUL IF SWALLOWED OR INHALED. If contacted on
skin immediately ush skin with plenty of water for at least 15 minutes. Remove
contaminated clothing and shoes. The dye stains pretty much everything, and can be
a real mess if spilled. Since it is a textile dye, if you get it on your clothes you will
need to learn to like blue polka dots!
PROCEDURE
Protein Standards you are provided with a standard stock solution of 700 mg/mL
Bovine Serum Albumin (BSA) and the Bradford reagent.
Using the stock solution, prepare six protein standards in duplicates (1 mL each)
containing 100, 200, 300, 500, 600, 700 g/m: BSA.
Note: Prepare the standards by serial dilution.
Use the same buffer (0.2 M phosphate buffer, pH 8) to prepare the standards and
the samplesto be assayed.
Assay Procedure
Set the spectrophotometer to measure a single wavelength 595 nm and an
Absorbance range of O to 2 Absorbance units. Use the same 4 mL plastic cuvette
for all measurements, starting with the lowest protein concentration and working up.
Shake out remaining drops between samples. Blank the spectrophotometer, using
the blank of (3 mL of Assay reagent and 0.04 mL distilled water). Assay each sample
by mixing 2.0 mL of Assay reagent and 0.04 mL of sample solution (if necessary,
dilute sample with assay buffer). Record the absorbance of the protein standards and
samples at 595 nm.

Lab report:
1) Prepare a graph of Absorbance at 595 nm vs [Protein] for the protein
standards.
2) Make sure the absorbance of each sample falls inside the linear range of the
standards.
3) Examine the graphed points and decide if any should be rejected. (Often a
single point can be rejected without invalidating the standard curve, but if
more than one point appears questionable the assay should be repeated.) The
Bradford assay gives a hyperbolic plot for absorbance vs protein
concentration, but within a range of relatively low protein concentrations, the
hyperbolic curve can be approximated reasonably well by a straight line. Use
a best-fit straight line to fit the points if you feel it will give a good fit. If not,
draw a smooth curve that falls on or near each of the data points
4) Determine the protein concentration of samples corresponding to their
absorbance using the standard curve
5) Comment on your results.
Experiment 3: Assay for -amylase activity in Saliva
Reaction of or -Amylase
The activity of -amylase enzyme can be assayed by determining the rate of
appearance of reducing sugars such as glucose or maltose (products) or by the rate
of disappearance of starch (substrate). Different types of assays can be used to
measure the appearance of products or is disappearance of the substrate.
Enzymatic Assay:
Two different ways to measure the above with -amylase.
1) Saccharogenic - This methodology measures the rate of appeqarance of
reducing sugars (glucose, maltose )
2) Amyloclastic - This method measures the hydrolysis of starch and the rate of
its disappearance;
Turbidometric Assay: Measures the disappearance of starch by measuring the
reduction in light scatter (due to the disappearance of starch).
Assay using Chromogenic substrates: A dyes bound to synthetic starch substrate,
with the dye being released (and measured) once the substrate is hydrolyzed.
Chromogenic substrate techniques are the current clinical amylase assay.
The enzyme activity assay procedure described in this practical is essentially that of
Somogyi, who rst quantied amylase activity by measuring the time required to
hydrolyze starch, in a carefully standardized substrate solution. A simple assay to
measure this time takes advantage of differently colored products generated by the
reaction between iodine and the saccharides depending of their degree of
degradation.
ot-Amylase on-Amylase
Starch (polysaccharide) .__..__> Oligosaccharides _---> Glucose + Maltose
I (mono/disaccharides)
[Blue with iodine] [Red with iodine] [Yellow with iodine]
After mixing the amylase-containing samples with a standardized starch solution,
the reaction is monitored by removing portions of the mixture at timed intervals
and adding these to aliquots of an iodine solution. As long as starch is present, a
blue-purplish color will develop. As the incubation proceeds, the color will change
from blue to blue-purple, to red-purple and then to reddish-brown.

The reaction is considered to have reached its endpoint when samples produce a
reddish-brown color with iodine. The time required to reach the endpoint is a
function of -amylase activity expressed in Somogyi units (one Somogyi Unit is
dened as the amount of amylase required to produce the equivalent of l mg of
glucose in free aldehyde groups in 30 minutes at 40 C. (Somogyi UniIS/dL may
be converted to International Units (pmol minute-1 L-1) by multiplying by 1.85.
[SalivaryAmylase] (Somogyi Units/dla) = Temperature Factor
Endpoint time [min]

The temperature factors for the incubation temperatures are:

Incubation Temperature Factor
Temperature (C)
37 18
40 16

Reagents:
Starch substrate - Soluble starch 0 75 g/L in 20 mM Tris-phosphate buffer, 10
mM NaCl, l mM NaF 0.1 mM NaN3, pH 7.0. (SHAKE WELL BEFORE USE) The
solution is slightly opalescent, and on standing threads or white sediment may
appear due to retrograded starch. This does not interfere with the assay.
Iodine solution - 0.2% iodine, containing 0.2 mM potassium iodide and buffer.
Dilute as necessary to give a blue color when mixed with the starch.
Dilutent solution - 0.5% NaCl solution

Assay Procedure:
1. Pipet 0.8 mL of iodine solution into each of 6 test tubes [100 x 75 mm]. Keep
tubes at room temperature.
2. Preparation of the reaction mixture:
- Swab the cap of the starch substrate bottle with ethanol.
- Using a needle and syringe, transfer l.5 mL of starch solution to the remaining t
L, est tube.
- Place the test tube in a heat block at 37 C and allow to incubate for 2 minutes.
- Add 0.1 mL of diluted saliva (Note 1) to the l.5 mL of starch substrate in the heat
block.
- Mix and record the starting time.
3. Aer 2 minutes, remove 0.20 mL of saliva-starch mixture from the test tub l '
e, eaving the remainder in
the heat block. Add this aliquot to one tube ofiodine solution Mix and record th t'
- e ime, color (Note 2).
4. After another 1 or 2 minutes withdraw a second 0.20 mL aliquot from the saliva
st h '
- arc mixture, and add
the aliquot to a second tube of iodine solution. Mix and record the time and color.
5. If the blue color persists, continue the incubation with sampling at regular
intervals until the reddish-
brownendpoint is observed.
1
6. Record the total elapsed time required to reach the endpoint.