Date Due: 9 Sept 2016

Date Submitted: 9 Sept 2016

EXPERIMENT NO. 3
MONITORING PROTEIN CONFORMATIONAL CHANGES BY VISCOSITY AND CD
SPECTROPSCOPY
Background of the Experiment
This experiment aims to be able to study the effects of various denaturants in crude protein extracts
through viscosity measurements and to assess circular dichroism spectra to determine the extent of denaturation.
Results and Discussion
Denaturation occurs in proteins when the bonding interactions in the secondary and tertiary structures are
disrupted. These bonding interactions include hydrogen bonding, salt bridges, disulfide bonds and non-polar
hydrophobic reactions so various reagents can cause denaturation of proteins.
Proteins can denature at either high or low extremes of pH. The addition of HCl or NaOH disrupt the
ionic bonds that hold salt bridges in the protein together. The positive and negative ions in the salt change
partners with the positive and negative ions in the acid or base added. The NaCl alters the ionic strength of the
solution, this would affect the ion bridges in its single organization. B-mercaptoethanol causes reduction of the
disulfide bridges to two sulfhydryl groups resulting to the complete disruption of the tertiary structure of the
protein. However, native conditions can still be recovered if experimental conditions are properly conducted.
Detergents such as sodium dodecyl sulfate (SDS) cause protein denaturation by disrupting the hydrophobic
interactions in the protein. Detergents are amphiphilic. The hydrophobic part of the detergent associate with the
hydrophobic parts of the protein and its hydrophilic ends interact with water causing the hydrophobic parts of the
protein to no longer associate with each other. Chaotropic agents like urea denature proteins by allowing water
molecules to solvate non-polar groups in the interior of proteins where water molecules disrupt the hydrophobic
interaction that would normally stabilize the native conformation.
Viscosity can be a good indicator to monitor protein unfolding. While the protein denatures, its protein
solubility decreases and the viscosity increases. The destruction of molecular interactions in the protein will
exhibit an increase in viscosity by occluding the liquid solution causing a higher resistance to flow. Denatured
proteins will provide longer flow rates. It means that the structure dominating the molecule is the strips and
stretched-out amino acids which are insoluble in water therefore making a more viscous solution. The relative
viscosity can be calculated by the equation below :

n
tp o
=
no to p
Where t = flow time
n/no= relative viscosity
In dilute solutions where pp0, the relative viscosity, nred, becomes t/to. The specific viscosity, nsp, is (t/to)
1. Therefore,

n =

n sp
c

Table 1. Viscosity Measurements

Denaturant
HCl
NaOH
Urea
BME
SDS
NaOH

Blank
(Nativ
e)
1.12
1.15
1.09
1.11
1.17
1.14

time (min.)
Native
Blank
albumi
(Denatur
n
e)
1.73
1.2
2.02
1.22
2.12
1.42
1.86
1.6
1.91
1.78
2.08
1.26

Denature
d albumin
2.12
2.5
3.12
4.16
5.12
2.65

Table 2. Calculated sp and red Values

Denaturant
Nsp Native

Nred Native

HCl
NaOH
Urea
Beta Mercaptoethanol
SDS
NaCl

54.46428571
75.65217391
94.49541284
67.56756757
63.24786325
82.45614035

0.5446428571
0.7565217391
0.9449541284
0.6756756757
0.6324786325
0.8245614035

Nsp
Denatured
0.7666666667
1.049180328
1.197183099
1.600000000
1.876404494
1.103174603

Nred denatured
76.66666667
104.9180328
119.7183099
160.0000000
187.6404494
110.3174603

For the denaturation of 1% albumin, Sodium Dodecyl Sulfate (SDS) is the denaturant with the highest
reduced viscosity with red=187.6404494. Based from the data, it can be concluded that the most effective
denaturant for 1% albumin is SDS followed by Beta Mercaptoethanol, Urea, NaCl, NaOH, and the least effective,
HCl.

Summary, Conclusion, and Recommendations

References
Horton, H. Robert. Principles of Biochemistry. Upper Saddle River, NJ: Prentice Hall, 1996.pp.104-106 Print.
2013. Biochemistry Laboratory Manual. Biochemistry Academic Group, Institute of Chemistry, UP Diliman.
Philippines pp. 23-24

Appendix
Native
HCl; native sample
sp = (t/t0) -1= (Native albumin/ Blank (Native))-1
= (1.73/1.12) -1
sp = 0.5446428571
red = sp/ c
= 0.5446428571/0.01
red = 54.46428571
NaOH; native sample
= (2.02/1.15) -1
sp = 0.7565217391
red = sp/ c
= 0.7565217391/0.01
red = 75.65217391
Urea; native sample
= (2.12/1.09) -1
sp = 0.9449541284
red = sp/ c
= 0.9449541284/0.01
red = 94.49541284
Beta Mercaptoethanol; native sample
= (1.86/1.11) -1
sp = 0.6756756757
red = sp/ c
= 0.6756756757/0.01
red = 67.56756757
SDS; native sample
= (1.91/1.17) -1
sp = 0.6324786325
red = sp/ c
= 0.6324786325/0.01
red = 63.24786325
NaCl; native sample
= (2.08/1.14) -1

sp = 0.8245614035
red = sp/ c
= 0.8245614035 /0.01
red = 82.45614035
Denatured
HCl; denatured sample
sp = (t/t0) -1= (denatured albumin/ Blank (Native))-1
= (2.12/1.2) -1
sp = 0.7666666667
red = sp/ c
= 0.7666666667 /0.01
red = 76.66666667
NaOH; denatured sample
= (2.5/1.22) -1
sp = 1.049180328
red = sp/ c
= 1.049180328 /0.01
red = 104.9180328
Urea; denatured sample
= (3.12/1.42) -1
sp = 1.197183099
red = sp/ c
= 1.197183099 /0.01
red = 119.7183099
Beta Mercaptoethanol; denatured sample
= (4.16/1.6) -1
sp = 1.600000000
red = sp/ c
= 1.600000000
red = 160.0000000
SDS; denatured sample
= (5.12/1.78) -1
sp = 1.876404494
red = sp/ c
= 1.876404494/0.01
red = 187.6404494
NaCl; denatured sample
= (2.65/1.26) -1
sp = 1.103174603
red = sp/ c

= 1.103174603/0.01
red = 110.3174603