1) What is the role of 0.

25M sucrose as the medium for the

fractionation process?
The purpose of the sucrose medium in the fractionation process
was to serve as a cushion, preventing the destruction of the
mitochondria and other organelles of the cell during the process
of fractionation also known as centrifugation.
2) List the major components that are present in (a) pellet P1, and
(b) supernatant S2.
In Pellet P1 you would find the nucleus and its contents while the
Supernatant S2 would contain all the other organelles excluding
mitochondria.
3) Define the Beer-Lambert Law and briefly explain why Absorbance
has no units.
Beer-Lamberts Law states that absorbance is proportional to
concentration and path length over a certain period of time
(A=(epsilon)(C)(L). Absorbance has no units because it is the
ratio of light passed through an object compared to the light that
is passed into it.
4) Plot a graph of absorbance vs. concentration for both sets of
dilutions (use the concentrations calculated from your dilutions,
do not use the concentrations obtained by using Beer-Lambert
law). Which dilution appears to be more accurate? Comment on
the spread of values.
Dilution
O
X2
X3
X4
X5

Concentration
0.30mM x 8ml = C2 x 40ml
C2 = 0.060mM
0.060mM x 2ml = C2 x 4ml
C2 = 0.030mM
0.060mM x 1ml = C2 x 3ml
C2 = 0.02mM
0.060mM x 1ml = C2 x 4ml
C2 = 0.015mM
0.060mM x 1ml = C2 x 5ml
C2 = 0.012mM

f(x) = 8.06x
Absorbance
(Graduated pipette)
Absorbance
(Automatic pipette)
Linear (Absorbance
(Automatic pipette))

The automatic pipette appears to have given the most accurate

reading. The Automatic pipette line is closest to the best-fit line.
The spread in values of the line formed by the automatic pipette
gives a better reading of the absorbance of visible light due to
the alkaline properties. The spread of values also indicate that as
the concentration increases the absorbance increases. Also as
the concentration increases the amount of light that it absorbs in
relation to the lower concentrations the disparity increasesl
5) Tabulate the data for the pH measurements for all solutions.
State whether the test solutions did or did not exhibit buffering
capacity. Explain your observations based on the composition of
the solutions.
Unknown A
Tube
1
2
3
4

pH before
7.7
7.6
7.0
6.9

pH after
2.76
8.6
6.9
7.08

Unknown B
Tube
1
2
3
4

pH before
7.07
7.07
5.48
5.48

pH after
2.51
11.15
5.11
5.60

Unknown C
Tube
1
2
3
4

pH before
7.2
7.1
6.3
6.3

pH after
2.4
11.3
5.8
7.8

Unknown D
Tube
1
2
3
4

pH before
7.2
7
6.7
6.8

pH after
2.8
9.2
5.6
6.0

Test tubes one and two for all Unknowns contained water. None
of them exhibited any buffering capacity as the pH changed
dramatically except for test tube 2 in unknown and the change
was still significant enough to state that it did not have buffering
capacity.
In unknown A test tubes 3 and 4 both exhibited buffering
capacity as the pH changed by only 0.1 and 0.18 units
respectively. This solution contained a weak acid and its
conjugate base and prevented a significant change in the pH
range.
In unknown B test tubes 3 and 4 also exhibited buffering
capacity, as the pH change was minimal. This solution contained
a weak acid and its conjugate base
In unknowns C and D test tubes 3 and 4 exhibited similar
properties to those of unknowns A and B, considering the
concentration and strength of the HCl and NaOH that was added
to the solution and the change in pH was not drastic at all.
6) List the major buffer systems in the blood of mammals and show
the equations for each system?
CarbonicAcid:BicarbonateBufferSystem
CO2+H2O H2CO3 H++HCO3
ProteinBufferSystem
Thereisnoequationforthissystem

HemoglobinBufferSystem
Thereisnoequationforthissystem
PhosphateBufferSystem
Na2HPO4+H+ NaH2PO4+Na+
7) Mention the importance of the Ninhydrin test in clinical or
research laboratory?
The importance of the Ninhydrin test is important in testing for
ammonia, amino acids, and other proteins or peptides. It is a
very sensitive test and can be used to test the amino acid
sequence of a protein. In research this test can be used to study
amino acids one by one as this test can identify them or separate
them for further analysis. It can also be used in research in
discovering new proteins if you find a new sequence of amino
acids. Clinically it is used to detect fingerprints. Also because it
can detect amino acid sequence, it allows for the detection of a
mutation in a protein rendering it unviable and a diagnosis can
be made.
8) Plot a graph of your standard curve for the Folin-Lowry assay.
Determine the concentration of your two unknowns and report
your answer in mg/ml.
Concentration
Blank
0.04
0.08
0.12
0.16
0.20
Unknown C
Unknown S

Absorbance
0.00
0.175
0.222
0.382
0.430
0.615
0.611
0.558

Absorbance

0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0.22
Absorbance

Concentration of unknown C = 0.611/2.72 =0.224mM

Concentration of unknown S = 0.558/2.72 = 0.205mM
9) Name the important chemical method available to determine
total protein in the body fluids?
This would be the Folin-Lowry assay method.
10)
What is denaturation of proteins? Mention a few denaturing
agents used in the laboratory?
Denaturation of a protein is the disruption of intermolecular
bonds that hold the secondary, tertiary, and quaternary (if
present) together. A few denaturing agents used in the lab are:
Extreme acidic conditions
Heat
Heavy metals: Lead, mercury,
Salinity
11)
What happens to the structural organization of protein
when it is denatured?
The secondary and tertiary structures are disrupted so the alpha
helix and beta-pleated sheets are no more and there is no 3Dimensional shape, there is only the linear shape because the
covalent peptide bonds in the primary structure are too strong to
break.

12)
Mention the importance of the denaturing process in the
research laboratory.
Denaturing of proteins firstly allows for proteins to be sorted out
exclusively by their molecular weight, and not being influenced
by the secondary structures.