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Graph 1

mmols of Titrant Added vs. pH observed


14.00
12.00
10.00

pH

8.00
6.00
4.00
2.00

-10.00

-5.00

0.00
0.00

5.00

10.00
15.00
mmols of Titrant

20.00

25.00

30.00

Estimate the pKa of the ionizable groups of histidine


At each equivalence pt, draw structure of ionized species that predominates at pH value

35.00

Table 3.
mmols of
titrant
needed
pH value
histidine
1.00
9.75
1.25
7.98
1.50
7.78
1.75
4.84
2.00
2.75
2.25
1.69
2.50
1.23
2.75
0.72
3.00
0.30
3.25
0.26
3.50
0.15
3.75
0.04
4.00
0.08
4.25
0.19
4.50
0.30
4.75
0.91
5.00
1.57
5.25
2.95
5.50
4.13
5.75
5.82
6.00
7.51
Sample Calculations:

water
0.30000
0.20714
0.13594
0.09677
0.05645
0.02477
0.02364
0.02251
0.02138
0.02024
0.01911
0.01798
0.01685
0.01572
0.01458
0.01345
0.01232

6.25
6.50
6.75
7.00
7.25
7.50
7.75
8.00
8.25
8.50
8.75
9.00
9.25
9.50
9.75
10.00
10.25
10.50
10.75
11.00
11.25
11.50
11.75
12.00
12.25
12.50

9.25
10.87
11.60
12.34
13.07
13.76
14.43
15.10
15.78
16.58
17.38
18.18
19.05
20.11
21.17
22.22
23.28
24.18
24.94
25.70
26.27
26.35
26.43
26.81
28.60 32.25 -

0.01119
0.01005
0.00892
0.00779
0.00666
0.00036
0.00262
0.00487
0.00713
0.00939
0.01164
0.01390
0.01616
0.01841
0.02067
0.02292
0.02568
0.03412
0.04257
0.05638
0.10957
0.17308
0.25000
0.41429

Graph 2.

pH value
14.00
12.00
10.00
8.00
pH value

6.00
4.00
2.00

-10.00

-5.00

0.00
0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

Graph 3.

Time vs. mmols of KOH


amount of KOH added (mmol)

0.25
0.2
0.15
0.1

mmols

0.05
0
0

10

15

20
time (min)

25

30

35

40

Results: The pH curve showed four flat lines, also the isoelectric points (pI). There is one at
around pH= 1.25, pH = 6.00, pH=10.25, and pH=12.25. The graph for Time vs. mmols of
KOH show an acute increase in KOH addition until around 10 minutes until it starts to
become stable.
Discussion:
From graph:
Use reaction endpoint to measure total mmoles of KOH needed to titrate protons
produced from BSA hydrolysis
Calculate the total number of peptide bonds cleaved
Since one mole of H+ will be released for every mole of peptide bond cleaved, if there is
10.95mmols of KOH used, then that must equal the concentration of H+s released.
So 3/2 peptide bonds per mol KOH = 3/2 peptide x mmol KOH = 3/2x 10.95mmol = 16.425
peptide bonds cleaved
Calculate the number of lysine + arginine residues per molecule of BSA (Mr is
66,000gm/mole)
Since trypsin cleaves after arginine and lysine residues, the number of residues would be
16 bonds.
So 3/2 peptide bonds per mol KOH = 3/2 peptide x mmol KOH = 3/2x 10.95mmol = 16.425
peptide bonds cleaved, this also should equal the number of lysine and arginine residues.

Assume pKa of the a-amino residues formed to be 8.2 and the pKa of the carboxyl
groups to be 3.5
C-1. What error in the number of arginine plus lysine in BSA is made by addition of
trypsin due to the solution in which trypsin is dissolved (0.001N HCl)
Since trypsin is in HCl, the extra H+ from the HCl can increase the number of H+ ions,
making it seem like the number of peptide bonds is more. Therefore, it may appear as if
there is an increased number of residues of lysine and arginine.