Course: Chem 271
Column Chromatography and Redox
Biochemistry
(Lab 3)
Laura LeClerc
Lab partner: Monica Treder
Student I.D.: 9764763
Experiment performed:
March 1st, 2011
Date Submitted: March 1st , 2011
Results
Table 1: LDH activity results
Fraction #
Colour
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Pink/brown
Pink/brown
Pink/brown
Pink/brown
Yellow
Yellow
Yellow
Yellow
-
Before Ascorbate
After addition of 25
uL of 1M Ascorbate
LDH activity %LDH activity
(ug/min)
0%
0%
0%
0%
-0.018
0.5%
-3.168
89.8%
-3.529
100%
-0.377
10.7%
-0.026
0.7%
-0.0123
0.3%
-0.015
0.4%
-
Absorbance of
Cytochrome c
(at  = 550 nm )
0.370
0.755
% change: 51.0 %
Enzyme
activity assay
stopped at
Fraction 10
due to time
constraints
Table 2:
Ascorbate
reduction
Absorbance of FMN (at 
= 450 nm )
0.682
0.665
% change: 2.5 %
Sample Calculations
Table 1: % LDH activity
Fraction 6: 0.377*100%/3.529 = 10.7%
Table 2: % change
FMN: 100 – [(100*0.665)/0.682] = 2.5%
Questions
Question 1: One would expect molecules to elute from a gel filtration chromatography
column in order of size, largest first to smallest last. This would be because in gel
filtration, the beads are made with indentations and inner cavities that trap and slow the
passage of smaller molecules, while larger molecules pass through unaffected by the
beads. Cytochrome C has a molecular weight of 13000 g/mol (1), LDH from rabbit
muscle has a molecular weight of 140000 g/mol but in a buffer pH 5 dissociates into two
70 000g/mol dimers (2), and FMN is 456.344 g/mol (3). Seeing as all three of them are
proteins one can assume that these vast differences in molecular weight can translate at
least vaguely to their relative sizes, so the expected order of elution would be LDH first,
then Cytochrome C, then FMN.
Question 2: The order in the fractions observed was LDH activity first (sample 5,)
cytochrome c’s reddish-brown colors (samples 7-10) and then FMN’s yellow colors
(samples 11-14). This is exactly as predicted.
Question 3: Without assaying LDH activity there would have been no way to detect
when the LDH eluted (which allows us to find out the order in which the proteins eluted).
It was also done to detect specifically in which samples LDH was present and where it
was most concentrated.
Question 4: The wavelength chosen for measuring cytochrome C reduction was  = 550
nm, and for FMN,  = 450 nm. For FMN,  = 450 nm is obviously the highest peak of
absorbance with the largest difference between the oxidized and reduced molecule’s
absorbance, making it optimal as a choice. For cytochrome C,  = 550 nm and  = 375
nm present approximately the same difference, however  = 550 nm is a far better
choice: this because there is also a very large peak for FMN’s difference between the
oxidized and reduced molecule at  = 375 nm, and this could come to interfere with the
discreeter results that cytochrome c’s analysis would yield seeing as its peaks (and
difference) are much smaller. This is why  = 550 nm, lying outside the range of
absorbance for FMN altogether, was chosen. While one could argue that there is a
possibility of affecting the results with impurities from cytochrome C when measuring
FMN’s change of absorbance because cytochrome C also has an absorbance difference
between oxidized and reduced states at  = 450 nm, this is a much smaller interference.
Question 5: At  = 550 nm, if Cytochrome c is reduced the absorbance should increase.
It did, and by a significant margin (increased by 51.0 %) so one could safely claim that it
has been reduced. At  = 450 nm, if FMN is reduced the absorbance should drop. It
did, but only by a small margin (2.5%) but the preparation of an ascorbate-added sample
and its measurement was repeated to verify the results, and exactly the same result was
yielded with unerring accuracy. One could attribute the change to a difference in the
material, use of equipment, impurities or a very small reduction having been achieved.
Question 6: At 400 nm, Cytochrome C’s measured absorbance would be much higher
than at 550 nm (perhaps between 1-2). However there should be little to no percieved
difference between the oxidized and reduced samples. At 400 nm, FMN still has a
reasonable difference between the oxidized and reduced forms, however seeing as at a
higher difference there was only a 2.5% drop in absorbance, the difference of
absorbances percieved before and after the ascorbate was added should have been
even smaller.
Bibliography
(1) J. Powlowski, M.J. Kornblatt, P. Joyce, J. Turnbull & Mihai Ciortea. (2010.)
“Laboratory & Tutorial Manual CHEM 271”. Laboratory 4, p.87.
(2) Worthington Biochemical Corporation. (2011.) <http://www.worthingtonbiochem.com/LDH/default.html>. Visited 15/03/2011.
(3) European Bioinformatics Institute. (2011.) <http://www.ebi.ac.uk/msdsrv/chempdb/cgi-bin/cgi.pl?FUNCTION=getByCode&CODE=FMN>. Visited 15/03/2011.