Part A Fractionation of amino acids

Table 1

Fraction No. Response to Ninhydrin test Response to Sakaguchi test

1 + -

2 + -

3 + -

4 - +

5 - +

6 - +

7 - +

Notes: “+” indicates positive result while “-” indicates negative result.

Part B Potentiometric titration of histidine

Mass of histidine = 0.3 g
MW of histidine= 6(12.011) + 9(1.008) + 3(14.007) + 2(15.999) =156.16 g/mol
0.3𝑔
No.of moles of histidine = 156.16 𝑔/𝑔𝑔𝑔= 1.921 x 10⁻³ mol = 0.001921 mol

0.001921
Volumes of 0.5 M HCL required = = 3.8 x 10⁻³ L x 1000 mL = 3.8 mL
0.5

𝑔𝑔𝑔𝑔𝑔 𝑔𝑔 𝑔𝑔𝑔𝑔 𝑔𝑔𝑔𝑔𝑔

Sample calculation of molar ratio: 𝑔𝑔𝑔𝑔𝑔 𝑔𝑔 ℎ𝑔𝑔𝑔𝑔𝑔𝑔𝑔𝑔

1
 0.1 𝑔𝑔𝑔/𝑔
Molar ratio for 0.5mL of NaOH = [(1000 𝑔𝑔/𝑔) x 0.5 mL] / (0.001921 mol) = 0.026

Table 2 Titration of Histidine

mLs NaOH Molar ratio (10−3 ) pH

0 0 4.65

0.5 0.026 4.66

1.0 0.052 4.67

1.5 0.078 4.68

2.0 0.104 4.71

2.5 0.13 4.75

3.0 0.156 4.78

3.5 0.182 4.83

4.0 0.208 4.87

4.5 0.234 4.90

5.0 0.26 4.94

5.5 0.286 4.98

6.0 0.312 5.00

6.5 0.338 5.03

7.0 0.364 5.06

2
7.5 0.39 5.09

8.0 0.416 5.11

8.5 0.442 5.13

9.0 0.468 5.16

9.5 0.494 5.19

10.0 0.52 5.21

10.5 0.546 5.25

11.0 0.572 5.28

11.5 0.598 5.31

12.0 0.624 5.34

12.5 0.65 5.41

13.0 0.676 5.43

13.5 0.702 5.45

14.0 0.729 5.49

14.5 0.754 5.53

15.0 0.781 5.55

15.5 0.807 5.61

16.0 0.833 5.65

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16.5 0.859 5.70

17.0 0.885 5.75

17.5 0.911 5.80

18.0 0.937 5.87

18.5 0.963 5.97

19.0 0.989 6.06

19.5 1.015 6.17

20.0 1.041 6.27

20.5 1.067 6.40

21.0 1.093 6.54

21.5 1.119 6.55

22.0 1.145 6.68

22.5 1.171 6.85

23.0 1.197 6.88

23.5 1.223 6.98

24.0 1.249 7.05

24.5 1.275 7.12

25.0 1.301 7.20

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25.5 1.327 7.27

26.0 1.353 7.36

26.5 1.379 7.42

27.0 1.406 7.48

27.5 1.432 7.56

28.0 1.458 7.62

28.5 1.484 7.70

29.0 1.51 7.79

29.5 1.536 7.83

30.0 1.562 7.89

30.5 1.588 7.95

31.0 1.614 8.03

31.5 1.64 8.10

32.0 1.666 8.13

32.5 1.692 8.20

33.0 1.718 8.24

33.5 1.744 8.30

34.0 1.77 8.34

5
34.5 1.796 8.39

35.0 1.822 8.40

35.5 1.848 8.43

36.0 1.874 8.48

36.5 1.9 8.54

37.0 1.926 8.58

37.5 1.952 8.63

38.0 1.978 8.68

38.5 2.004 8.73

39.0 2.03 8.77

39.5 2.056 8.84

40.0 2.082 8.89

40.5 2.108 8.95

41.0 2.134 9.09

41.5 2.16 9.16

42.0 2.186 9.22

42.5 2.212 9.30

43.0 2.238 9.39

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43.5 2.264 9.49

44.0 2.29 9.59

44.5 2.316 9.69

45.0 2.342 9.81

45.5 2.368 9.93

46.0 2.394 10.03

46.5 2.421 10.15

47.0 2.446 10.26

47.5 2.472 10.32

48.0 2.498 10.41

48.5 2.524 10.48

49.0 2.55 10.56

49.5 2.577 10.63

50.0 2.603 10.66

50.5 2.629 10.70

51.0 2.655 10.74

51.5 2.681 10.78

52.0 2.707 10.84

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52.5 2.733 10.86

53.0 2.759 10.89

53.5 2.785 10.92

54.0 2.811 10.94

54.5 2.837 10.96

55.0 2.863 10.99

55.5 2.889 11.00

56.0 2.915 11.04

56.5 2.941 11.06

57.0 2.967 11.08

57.5 2.993 11.10

58.0 3.019 11.11

58.5 3.045 11.15

59.0 3.071 11.17

59.5 3.097 11.18

60.0 3.123 11.20

60.5 3.149 11.21

61.0 3.175 11.22

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61.5 3.201 11.23

62.0 3.227 11.25

62.5 3.253 11.26

63.0 3.279 11.28

63.5 3.305 11.29

64.0 3.331 11.30

64.5 3.357 11.31

65.0 3.383 11.32

65.5 3.41 11.33

66.0 3.436 11.34

66.5 3.461 11.35

67.0 3.488 11.36

67.5 3.513 11.37

68.0 3.54 11.38

68.5 3.566 11.38

69.0 3.591 11.39

69.5 3.618 11.40

70.0 3.644 11.41

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 70.5 3.67 11.42

71.0 3.696 11.43

71.5 3.722 11.43

72.0 3.748 11.44

72.5 3.774 11.45

73.0 3.8 11.46

73.5 3.826 11.46

74.0 3.852 11.47

74.5 3.878 11.48

75.0 3.904 11.49

75.5 3.930 11.50

76.0 3.956 11.52

76.5 3.9823 11.53

77.0 4.008 11.54

77.5 4.034 11.55

78.0 4.060 11.55

78.5 4.086 11.55

Figure 1 Titration curve

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Questions and Discussion

Part A

1. Glycine is firstly eluted from the column while arginine is the last. According to the results
from Ninhydrin test, the first three fractions show positive results, the presence of blue-purple
colour, which means there is presence of glycine in the first three fractions. The Ninhydrin Test
is a test for amino acids and proteins with a free -NH2 group. In Sakaguchi test, the last four
fractions show positive results, the presence of yellow-orange colour, which means there is
presence of arginine in the last four fractions. The elution rate of the amino acid is based on the
resin used. Dowex 50 is used in this ion exchange chromatography, it is a strongly acidic
polystyrene resin with negative charged (Campbell and Farrell 2014). Therefore, amino acids
that have a net charge opposite to that of the exchanger stick to the column and exchanging
places with the bound counterions, while the amino acids that have no charge or have the same
charge as the exchanger will elute first. In this case, glycine will flow freely through the resin

11
and eluted from the column faster as it is an amino acid with no net charge. Whereas arginine
will stay longer in the column because it has net positive charge which is opposite charged to the
Dowex 50 resin.

2. The Sakaguchi test is a test for the arginine. Arginine has a positively charged guanidine group
as the side chain. It is a compound with the formula of HNC(NH2)2. The positively charged
guanidine group attached to the arginine is stabilized by resonance structures between the two
NH2 groups (Bhagavan and Chung 2015). Therefore, guanidine group is the specific structure on
the arginine which associated with Sakaguchi test. This side chain present in arginine reacts with
α-Naphthol and alkaline hypobromite to give red-coloured complex (Dandekar and Rane 2004),
which show positive results in Sakaguchi test.

3. Write down structures which show the predominant ionic form of lysine at pH 1.0, 6.0, 9.5
and 11.0.

At pH 1.0, both the NH3+ are predominating, HO2CCH(NH3+)(CH2)4NH3+

At pH 6.0, the predominant ionic form is -O2CCH(NH3+)(CH2)4 NH3+
At pH 9.5, the predominant ionic form is -O2CCH(NH2)(CH2)4 NH3+
At pH 11.0, the predominant ionic form is -O2CCH(NH2)(CH2)4NH2

Part B

1. What are the pKa values of histidine? (This point will be the pH of ½ neutralization.)
9.17

2. What is the isoelectric point for histidine?

The isoelectric for histidine is calculated by first identifying the isoelectric form (that has a net
6.37+10.63
charge of zero) of the amino acid. Isoelectric point (pI) of histidine is = 8.5 (Champe
2

and Harvey 1994).

12
3. How many pKa values would you expect for histidine and what are their values as reported in
the literature?

Histidine consists of three acidic groups which are carboxylic acid with pKa=1.82, pyrrole NH
with pKa=6.04, and ammonium NH with pKa=9.17.(Campbell and Smith 1994) Histidine can be
having four forms which are three extra protons(a), two extra protons(b), one extra proton(c) or
completely deprotonated(d). Any of these forms relied on the pH of the solution, from pH of acid
to base. When we add base, COOH will be removed first because it is the most acidic proton,
follow by pyrrole NH then lastly the amino NH. It happened in each forms.

a is the dominant form at pH < 1.82

b is the dominant form in the range 1.82 < pH < 6.02
c is the dominant form, and in the range 6.02 < pH < 9.17
d is the major form in solution when pH > 9.17

Conclusion

This experiment showed that glycine and arginine are positive to ninhydrin test which
Ruhemann’s purple product will formed. Glycine eluted out first before the arginine because
presence of primary amine group. Guanidine group obtained in arginine in all fractions are pink
colour solution except the first and the second fractions.The isoelectric point for histidine is 8.5.

Reference

13
Bhagavan, N.V. and Chung, E.H., 2015. Essentials of Medical Biochemistry: With
Clinical Cases, 2nd ed. USA: Academic Press.

Campbell, M. K. and Farrell, S. O., 2014. Biochemistry. USA: Cengage Learning.

Campbell, P.N. and Smith, A.D., 1994. Biochemistry Illustrated. 3rd Ed. New York: Longman
Group UK.

Champe, P.C. and Harvey, R.A., 1994. Biochemistry. 2nd Ed. USA: J.B. Lippincott Company.

Dandekar, S.P. and Rane, S.A., 2004. Practical And Viva In Medical Biochemistry. India:
Elsevier.

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