COURSE CODE: BIOL 2365 Comparative Biochemistry
TITLE: Proteins and Amino Acids
Table 1: The results of experiment 1; the Lowry Test
Volume of Standard Protein/ Unknown (mL)
Absorbance at 750 nm
1.0 Unknown 1a
1.0 Unknown 1b
1.0 Unknown 2a
1.0 Unknown 2b
Table 2: The results of Experiment 2; Ninhydrin Test
Table 3: The results of Experiment 3; Protein Precipitation
White fluffy precipitate in large mass, the fluffy mass goes from the bottom to the top of the tube. The solution was thin and loose. Ammonium sulphate
White gel-like precipitate, the solution was thick.
White precipitate accumulated at the top and also the bottom of the solution. The solution was thin. NaOH
No precipitate seen, the solution was cream in color.
Light blue coagulated precipitate seen at the top of the solution. Lead Acetate
Cloudy solution with small specks of white precipitate.
Ice cold ethanol
Cloudy at the bottom and about 0.5 cm of the top of the solution was clear.
Table 4: The results of Gel Filtration Chromatography
Volume collected in mL
Clear phosphate buffer
Table 5: The result of Electrophoresis of Amino Acids
Distance moved (+/-) cm Amino acid
10% Acetic acid
(1) Protein concentration of Standard Protein Solution:
1mL = 200µg protein
Therefore 0.1mL= 0.1/1× 200 = 20µg
(2) Protein concentration of the two unknowns:
Gradient of the line = Y2-Y1/ X2-X1
0.135-0.017/ 60-20 = 0.00295
Average absorbance of Unknown 1= 0.463 + 0.433/ 2 = 0.448
Therefore: 0.448 = 0.00295 x
x = 0.448/ 0.00295 = 151.86µg
Therefore 1mL of Unknown 1 has 151.86µg of protein
Average absorbance of Unknown 2 = 0.237 + 0.159/ 2 = 0.198
Therefore: 0.198 = 0.00295 x
x = 0.198/ 0.00295 = 67.12µg
Therefore 1mL of Unknown 2 has 67.12µg of protein
(3) The void volume is the volume between and outside the gel beads in the column. This volume is thus calculated using the eluate value of the largest molecule (macromolecule) that was completely excluded from the column. This is so since the largest molecule occupies the spaces between and outside the gel beads since they are too large to enter and thus they travel out more quickly than the smaller molecules which can become trapped in the pores of the beads. Therefore the total volume of this particle will indicate the void volume. The macromolecule in this experiment was blue dextran since it was eluated first. The amount of blue dextran was 3.00mL; therefore the void volume was also 3.00mL. (4) Bed volume = volume of a cylinder = π r2h
Where h = 15cm r = 0.5cm and π = 3.14
Bed volume = 11.78 cm3
(5) The fraction of bed volume that is void volume = 3.00/ 11.78 = 0.254 (25.4%)
The Lowry Assay is a biochemical test that assists in the determination of the total amount of protein present in a solution. The principle of this assay is grounded on the reactivity of the peptide nitrogen with copper ions in alkaline conditions followed by a reduction of the Folin- Ciocalteu’s phosphomolybdiphosphotungstic acid; which is yellow in color to heteropolymolybdenum which is blue in color. This is done via an oxidation of aromatic protein residues by copper (Boyer 2012). The reaction is as follows:
Figure 1: the Lowry reaction
A spectrophotometer was used to measure the color change of the Folin- Ciocalteu’s reagent; this absorbance value is then valuable in determining...
References: Amrita University. Qualitative Analysis of Amino Acids.2012. http://amrita.vlab.co.in/?sub=3&brch=63&sim=1094&cnt=1 (4 October 2012)
Boyer, Rodney. 2012. Biochemistry Laboratory. Pentice Hall Publishers, New Jersey.
Nelson, Cox. 2008. Leningher Principles of Biochemistry. Fifth edition. W. H Freeman and Company, New York.
Nigam Aarti, Ayyahguri.2012. Archana Lab Manual in Biochemistry, Immunology and Biotechnology. Tata Mc Graw Hill Publishing Company.
pHscale. Net, pKa and Ka, 2012. http://www.phscale.net/pka-ka.htm (13 October 2012).
University of California, Department of Chemistry. 2011. Chemistry 2B Laboratory Manual. University of California. Davis, CA.
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