EXPERIMENT OF SPECTROPHOTOMETRY (PROTEIN)

INTRODUCTION

Spectrophotometry is a procedure that is frequently utilized in biological

laboratories. Probably the most common application in biology of this technique is
in the measurement of the concentration of a compound in solution. Using a
spectrophotometer, which measures the absorption by a solution of light of specific
wavelengths (visible or not), allows us to determine concentration such as protein
concentration. A second application of spectrophotomerty is the determination of
the absorption spectrum of a compound. (Both of these can be applied to colourless
as well as coloured solutions since a spectrophotometer can measure absorbance of
light that we cannot see.)
In this experiment, Biuret assay are used biuret reagent to detect the
presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-
colored coordination complexes in an alkaline solution. Several variants on the test
have been developed, such as the BCA test. The Biuret reaction can be used to
assess the concentration of proteins because peptide bonds occur with the same
frequency per amino acid in the peptide. The intensity of the color, and hence the
absorption at 550 nm, is directly proportional to the protein concentration,
according to the Beer-Lambert law. Despite its name, the reagent does not in fact
contain biuret ((H2N-CO-)2NH). The test is so named because it also gives a positive
reaction to the peptide-like bonds in the biuret molecule.

OBJECTIVES

1) To determine protein concentration in various type of protein content.

2) To determine protein concentration using protein assays which is Biuret
assay.
3) To learn how to use spectrophotometer in measuring the absorbance of
different

MATERIALS

7 test tubes
Tube rack
Deionized water (dH2O)
Micropipette
Biuret reagent
Spectrophotometer
Titter fish
Cencaru fish
METHODOLOGY

The steps that involves in this experiment are firstly, we are used 7 test
tubes; each test tube is filled with different volume of water, protein concentration
and volume of gelatin. The test tubes are labelled as a tube 1, 2, 3, 4, 5, 6 and 7.
The first test tube containing only distilled water that acts as a references or
control. The dilution was done by bringing the total volume to 1.00 mL with
distilled water and gelatin. Then, 2 mL of Biuret reagent are added to each tube
being vortex immediately after that. The solutions were left for about 15 minutes to
incubate it. To determine the absorbance of these solutions, a small amount of
each solution was transferred into cuvette and gently wipes the cuvette with a
paper towel to remove fingerprints and dust. Then, the absorbance readings were
taken. Using the concentration data and absorbance data, a standard curve can be
constructed, which is presented as the graph of absorbance vs concentration.

In the second part of this experiment, two different samples which are titter
fish and cencaru fish were prepared by extract the samples to get the solution.
Firstly, we take the mass of the samples which is both of the samples is 0.69 g.
then, we added 12 mL of distilled water to each sample for dilute it. Each solution
was then being added with Biuret reagent and immediately vortex. Then, incubate
the solution for about 15 minutes. Next, the absorption was recorded using a
spectrophotometer. Finally, the concentrations of the unknowns were estimated
using the standard curve plotted using the data from the first part of the
experiment.

RESULTS

Test Gelatin, H2O, Concentration 2mL of 15 Absorbance

tube mL mL protein, Biuret minutes
mg/mL incubation
1 0 1 0 0.000
2 0.1 0.9 1 0.031
3 0.2 0.8 2 0.082
4 0.3 0.7 3 0.086
5 0.4 0.6 4 0.117
6 0.5 0.5 5 0.291
7 0.6 0.4 6 0.262
8 1 mL of titter fish 1.25 0.0591
9 1 mL of cencaru 4.25 0.185
fish
Table 1: Result for the protein solution
 Figure 1: 7 test tubes that carry out in this experiment

absorbance Absorbance vs protein concentration

0.35

0.3

0.25

0.2

0.15

0.1

0.05

X1 = 1.25 X 2= 4.25
0
0 1 2 3 4 5 6 7
protein concentration, mg/mL

Graph1: graph for absorbance versus protein concentration

X1 = protein concentration for titter fish

X2 = protein concentration for cencaru fish

Based on the graph, the concentration for titter and cencaru fish is:

X1 = 1.25

X2 = 4.25

DISCUSSIONS

From this experiment, we want to determine the absorbance of the protein

and the unknown concentration for some sample of protein. So, we are using
spectrophotometric machine to get the value of absorbance for all the solution that
we used in this experiment.

From the data that we got, we can plot a graph of absorbance against
protein concentration. The graph that we got is absorbance is linearly proportional
to protein concentration. Then, from the graph also we can know the concentration
of the sample protein which is titter fish and cencaru fish. The concentration of
titter fish is 1.25 with the absorbance value is 0.0591 while for the cencaru fish is
4.25 with absorbance value is 0.185. What can we say here, the cencaru fish
contain high protein while titter fish contain low protein. So, we can suggest the
fish that has high concentration is cencaru fish. It will help to maintain and lose
weight, at the same time, it also works to stabilize blood sugar levels, improve
ability to learn and concentrate, reduce brain fog, boost energy levels, support
muscles and bones and support the absorption of important nutrients.

Next, A few possible errors can happen in this lab such as using the same
micropippete tip for both the distilled water and the gelatin. Another possible error
that could occur would be forgetting to blank the spectrophotometric and also
forgetting to wipe the fingerprints off of the cuvettes. If this error happened in this
experiment, the spectrophotometric cannot read the absorbance of the solution. So,
we should take some precaution when carry out this experiment to get the best
result.

CONCLUSIONS

In conclusion, by finding the absorbance and concentration levels of both

the known and unknown solutions evaluated in this experiment, the unknown
concentration was able to be determined. This experiment could be extended
further by repeating the lab with different solutions to help identify any unknown
solution.

REFERENCES

https://www.scribd.com/doc/41425746/Protein-Lab-Report-Experiment-3

http://biochemistrygirls.blogspot.my/2013/04/experiment-2-protein-
experiment.html