David Anthony (CID: 01520961)

A630 of bromocresol green at different concentrations of Albumin

A630 corrected for
background
A630 measured absorbance

The Average A630 Standard

concentration of corrected for Deviation of A630 /
Albumin Concentration of background corrected Calculated [Albumin]
(mg ml-1) Albumin (μM) absorbance absorbances A630 Values (μM-1)
1 2 3 1 2 3
0.0 0.00 0.108 0.110 0.124 0.000
0.1 1.52 0.184 0.191 0.202 0.076 0.081 0.078 0.078 0.00252 0.183 0.052
0.2 3.03 0.325 0.302 0.343 0.217 0.192 0.219 0.209 0.01504 0.314 0.069
0.3 4.55 0.460 0.462 0.479 0.352 0.352 0.355 0.353 0.00173 0.411 0.078
0.4 6.06 0.565 0.565 0.550 0.457 0.455 0.426 0.446 0.01735 0.487 0.074
0.5 7.58 0.631 0.650 0.692 0.523 0.540 0.568 0.544 0.02272 0.548 0.072
1.0 15.15 0.964 0.971 1.030 0.856 0.861 0.906 0.874 0.02754 0.729 0.058
2.0 30.30 1.091 1.076 1.186 0.983 0.966 1.062 1.004 0.05123 0.874 0.033
3.0 45.45 1.123 1.137 1.216 1.015 1.027 1.092 1.045 0.04143 0.936 0.023
4.0 60.61 1.127 1.157 1.236 1.019 1.047 1.112 1.059 0.04771 0.970 0.017
5.0 75.76 1.164 1.183 1.273 1.056 1.073 1.149 1.093 0.04952 0.992 0.014
David Anthony (CID: 01520961)

How A630 of bromocresol green varies with Albumin concentration

1.2

Amax = 1.09

0.8
A630

0.6

0.4

0.2

KD = 7.5
0
0 10 20 30 40 50 60 70 80
Concentration of Albumin (μM)
Average absorbance corrected for background absorbance Calculated A630 Values

How A 630 varies with A630/[P] for analysis of linear response equation
1.2
y = -12.34x + 1.2745,
where y is A630 and x is
A630/[P].
Therefore, KD = 12.34,
1 and Amax = 1.2745

0.8
A630

0.6

0.4

0.2

0
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
A630/[P]
David Anthony (CID: 01520961)

A450 of bromocresol green at different concentrations of Albumin

A450 corrected for
background
A450 measured absorbance

The Average A450 Standard

concentration of corrected for Deviation of
Albumin Concentration of background corrected
(mg/ml) Albumin (μM) 1 2 3 1 2 3 absorbance absorbances

0.0 0.00 1.100 1.118 1.216

0.1 1.52 1.068 1.073 1.188 -0.032 -0.045 -0.028 -0.035 0.00889
0.2 3.03 1.033 1.066 1.147 -0.067 -0.052 -0.069 -0.063 0.00929
0.3 4.55 0.997 0.993 1.084 -0.103 -0.125 -0.132 -0.120 0.01513
0.4 6.06 0.950 0.948 0.974 -0.150 -0.170 -0.242 -0.187 0.04839
0.5 7.58 0.927 0.932 1.039 -0.173 -0.186 -0.177 -0.179 0.00666
1.0 15.15 0.786 0.798 0.869 -0.314 -0.320 -0.347 -0.327 0.01758
2.0 30.30 0.733 0.707 0.820 -0.367 -0.411 -0.396 -0.391 0.02237
3.0 45.45 0.692 0.703 0.774 -0.408 -0.415 -0.442 -0.422 0.01795
4.0 60.61 0.684 0.695 0.744 -0.416 -0.423 -0.472 -0.437 0.03051
5.0 75.76 0.700 0.705 0.764 -0.400 -0.413 -0.452 -0.422 0.02706

How A450 of bromocresol green varies with Albumin concentration

Concentration of Albumin (μM)
0 10 20 30 40 50 60 70 80
0
KD = 9
-0.05

-0.1

-0.15

-0.2
A450

-0.25

-0.3

-0.35

-0.4
Amax = -0.44
-0.45

-0.5
David Anthony (CID: 01520961)

Exercise 1:
Both curves have roughly the same shape, however, they start and plateau at different values. The
reason the values are so far apart from each other is more likely due to the equation incorrectly
modelling the situation. In using that equation to model the situation, the assumption is made that,
because this is an example of weak binding, the free protein concentration is roughly equal to the
total protein concentration, i.e. that the concentration of the protein ligand complex is zero. In
reality, the concentration of the protein ligand complex is not zero, so the concentration of the free
protein will be less than that of the total protein concentration. When looking at the equation used
to model the calculated values, if [P] decreases, then the calculated values have a steeper
gradient, as my values suggest. Therefore, the concentration of the ligand protein complex
becomes more significant at higher protein concentrations. The greater the protein concentration,
the more the equilibrium shifts towards producing complexes until saturation is reached, so [PL]
can’t be assumed to be zero in the range at which I’m looking.

Exercise 2:
For graph 1, my KD value was 7.5 and my Amax value was 1.09, and for graph 2, K D was 12.34 and
Amax, 1.27. The first method for working out K D and Amax is going to be more accurate. This is
because Amax is taken straight from the data obtained (though there may be some error associated
with locating the asymptote) and then the equation says that when K D = [P], A360 = Amax/2, which
has not assumed that [P] = [P] 0 (which was the problem associated with the first question).
However, the second method uses a rearrangement of the original equation, such that the graph
produced would give a linear response. From the graph, it is clear that said response is not linear
(though a straight line has been drawn anyway). This again shows that the original equation does
not completely model what is occurring, especially at higher concentrations of protein. The K D and
Amax have been taken from the equation of the straight line drawn as K D = the gradient, and Amax =
the y-intercept. For these reasons, I believe the values obtained from the first graph are more
accurate.

Exercise 3:
For the A630 curve, at low concentrations of albumin, the absorbance should be low as there are
very few protein ligand complexes (what is actually absorbing at this wavelength) at this point.
Then as [P] increases, more and more complexes form to a point where the dye molecules start to
become limiting and the absorbance plateaus with the maximum number of complexes produced.
For the A450 curve, the opposite is expected. At low concentrations of protein, absorbance of the
dye particles should be great (with the maximum occurring when there is no protein present). Then
the absorbance should decrease as more and more dye particles get bound to proteins in
complexes. Eventually all dye particles get bound and like the above graph, it should plateau. Both
graphs reflect this so qualitatively, they are consistent. Note all the absorbances are negative in
the second graphs as they have been corrected for background absorbance, i.e. all values on the
graph have had the maximum absorbance (from a solution of only ligand) taken away.
Quantitatively, the point at which A 630 would be at its maximum on the second graph is when the
graph plateaus for which the absorbance is -0.44. This leads to a K D of 9 using the same method
as for graph 1, which gave a K D of 7.5. So I would say that both graphs are consistent with each
other.