E3 Protein Denaturation

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 pàp0, 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 ηsp and ηred 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 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

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

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

= 1.103174603/0.01 ηred = 110.3174603