E3 Protein Denaturation

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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

n o

=

tp o

t o p

Where t = flow time

n/no= relative viscosity

In dilute solutions where pàp0, the relative viscosity, nred, becomes t/to. The specific viscosity, nsp, is (t/to)

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n

¿=

n sp

c

Table 1. Viscosity Measurements

Table 2. Calculated ηsp andηred Values

For the denaturation of 1% albumin, Sodium Dodecyl Sulfate (SDS) is the denaturant with the highest reduced viscosity with ηred=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 time (min.) Denaturant Blank (Nativ e) Native albumi n Blank (Denatur e) Denature d albumin HCl 1.12 1.73 1.2 2.12 NaOH 1.15 2.02 1.22 2.5 Urea 1.09 2.12 1.42 3.12 BME 1.11 1.86 1.6 4.16 SDS 1.17 1.91 1.78 5.12 NaOH 1.14 2.08 1.26 2.65

Denaturant Nsp Native Nred Native Nsp

Denatured Nred denatured HCl 0.5446428571 54.46428571 0.7666666667 76.66666667 NaOH 0.7565217391 75.65217391 1.049180328 104.9180328 Urea 0.9449541284 94.49541284 1.197183099 119.7183099 Beta Mercaptoethanol 0.6756756757 67.56756757 1.600000000 160.0000000 SDS 0.6324786325 63.24786325 1.876404494 187.6404494 NaCl 0.8245614035 82.45614035 1.103174603 110.3174603

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Appendix Native

HCl; native sample

ηsp = (t/t0) -1= (Native albumin/ Blank (Native))-1 = (1.73/1.12) -1

ηsp = 0.5446428571 ηred = ηsp/ c

= 0.5446428571/0.01 ηred = 54.46428571 NaOH; native sample = (2.02/1.15) -1 ηsp = 0.7565217391 ηred = ηsp/ c

= 0.7565217391/0.01 ηred = 75.65217391 Urea; native sample = (2.12/1.09) -1 ηsp = 0.9449541284 ηred = ηsp/ c

= 0.9449541284/0.01 ηred = 94.49541284

Beta Mercaptoethanol; native sample = (1.86/1.11) -1 ηsp = 0.6756756757 ηred = ηsp/ c = 0.6756756757/0.01 ηred = 67.56756757 SDS; native sample = (1.91/1.17) -1 ηsp = 0.6324786325 ηred = ηsp/ c = 0.6324786325/0.01 ηred = 63.24786325 NaCl; native sample = (2.08/1.14) -1

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ηsp = 0.8245614035 ηred = ηsp/ c = 0.8245614035 /0.01 ηred = 82.45614035 Denatured HCl; denatured sample

ηsp = (t/t0) -1= (denatured albumin/ Blank (Native))-1 = (2.12/1.2) -1

ηsp = 0.7666666667 ηred = ηsp/ c

= 0.7666666667 /0.01 ηred = 76.66666667 NaOH; denatured sample = (2.5/1.22) -1 ηsp = 1.049180328 ηred = ηsp/ c

= 1.049180328 /0.01 ηred = 104.9180328 Urea; denatured sample = (3.12/1.42) -1 ηsp = 1.197183099 ηred = ηsp/ c

= 1.197183099 /0.01 ηred = 119.7183099

Beta Mercaptoethanol; denatured sample = (4.16/1.6) -1 ηsp = 1.600000000 ηred = ηsp/ c = 1.600000000 ηred = 160.0000000 SDS; denatured sample = (5.12/1.78) -1 ηsp = 1.876404494 ηred = ηsp/ c = 1.876404494/0.01 ηred = 187.6404494 NaCl; denatured sample = (2.65/1.26) -1 ηsp = 1.103174603 ηred = ηsp/ c

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= 1.103174603/0.01 ηred = 110.3174603

Figure

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References