Biochemistry

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Calculation of the pH of test tube 1 using Henderson-Hasselbalch equation:

pKa of acetic acid = 4.7

Henderson-Hasselbalch equation: pH = pKa + [pic 6]

pH = pKa + [pic 7]

Mol of sodium acetate, n = [pic 8]

= [pic 9]

= 1 x 10-4 mol mol[pic 10]

Assume that Henderson-Hasselbalch equation equation will be:

pH = pKa + [pic 11]

pH = pKa + log [pic 12]

pH = pKa + log [pic 13]

The expected pH for Test tube 1:

Mol of acetic acid, n = [pic 14]

= [pic 15]

= 1 x 10-5 mol mol[pic 16]

pH = pKa + log [pic 17]

= 4.7 + log [pic 18]

= 4.7 + 1

= 5.7

Thus, the expected pH for test tube 1 is pH 5.7.

Questions & Answers:

- The optimum pH for most biochemical experiments is between the ranges of pH 6 – 8. The optimal buffering range for a buffer is the dissociation constant of the weak acid component of the buffer (pKa) plus or minus pH unit (Shintani & Polonský, 1997). Based on table 3, the pH reading increased gradually for both pH 6.0 and pH 7.0. pH 8.0 shown the largest differences, which is 1.78 after the addition of NaOH. Phosphate buffer at pH 7.0 has shown the smallest changes, which is 0.23. The increasing of pH value is due to increased of buffering capacity while shifting towards pKa value. The good buffering capacity for tris buffer is at highe pH, which is pH 8.0. A buffer is more effective when the pH is nearer pKa value (Moran, Horton, Scrimgeour & Perry, 2012). Thus, phosphate buffer, pH 7.0 and tris buffer pH 8.0 are best used for buffer performance.

- Table 5 indicates the ΔpH for phosphate buffer pH 7.0 at ice, room temperature (27°C), 50°C, 70°C is 0.03, 0.23, 0.15, 0.19 respectively and the ΔpH for tris buffer, pH 8.0 at ice, room temperature (27°C), 50°C, 70°C is 0.03, 1.78, 0.38, 0.73 respectively. Both pH of phosphate buffer and tris buffer change with temperature, but the changes in phosphate buffer only a little compared to tris buffer. pH is influenced by temperature due to that a temperature-dependent change to the dissociation of ions in a solution. The experiment should result in increasing ΔpH for both buffers at all temperature showing a decrease in buffering capacity (Boyer, 2009). Increasing in temperature caused a slight decrease in pH. This is because rise in temperature is associated with increased molecular vibration. Thus, when temperature increased, concentration of observable hydrogen ions also increased due to the tendency of forming hydrogen bonds decreased and lead to the reduce of pH value.

Table 7 indicates that ΔpH for phosphate buffer at dilution 1/10, 1/50, 1/100 with 4.34, 4.10, 3.54 respectively. Table 8 indicates that ΔpH for tris buffer at dilution 1/10, 1/50, 1/100 with 3.02, 3.01, 3.47 respectively. Dilution to buffer affects its pH value because pH varies according to the concentration of buffer ions. An increase in dilution factor reduces the amount of active buffer ions thus lowering buffering activity.

- Changes in temperature or dilution are expected to affect the buffering capacity. It is consistent with the observations. pH is influenced by both temperature and dilution. When temperature increased, there is a slight decrease in pH in both phosphate buffer and tris buffer. As the dilution factor increased, the pH value decreased. Tris buffer is more effective than phosphate buffer as tris buffer’s pH is nearer to the pka value hence having higher buffering capacity.

- Isoelectric point (pI) of casein is pH 4.6. Casein is the most important protein in milk. Acidification is used to isolated casein from milk by bring it to its isoelectric point. At the isoelectric point, the number of positive chargeson a protein equals the number of negative charges (Bettelheim & Landesberg, 2010).

- The isoelectric point of a protein is the pH at which protein is electrically neutral. At this pH, the overall charge on the protein is zero. The number of positive charges on the protein is equal to the number of negative charges, At the isoelectric point, the protein molecules usually precipitate because they do not carry a net charge. The viscosity of solubilized protein and the swelling power of insoluble proteins are at a minimum at the isoelectric point (Belits, Grosch & Schieberle. 2009). In the experiment, maximum precipitation is found in test tube 4 proving that it’s the isoelectric point. test tube 1 has no precipitation proving higher solubility away from isoelectric point and minimum solubility at isoelectric point. Test tube 1 has no precipitation proving higher solubility away from isoelectric point and minimum solubility at isoelectric point.

References

Belitz, HD, Grosch, W & Schieberle, P 2009, Dissociation, Food Chemistry, Springer, Germany, pp. 58-59.

Bettelheim, FA & Landerberg, JM 2010, Isolation and Identification of Casein, Laboratory Experiments for Introduction to General, Organic and Biochemistry, Brooks/Cole, Cengage Learning, Canada, pp. 477-488.

Boyer, R 2009, Biochemistry Laboratory: Modern Theory and Techniques, Pearson Benjamin Cummings, USA, pp. 374-376.

Moran LA, Horton HR, Scrimgeour KG & Perry MD 2012, Proteins: Three-Dimensional Structure and Function, Principles of Biochemistry 5th Edition. Pearson Education, USA, pp. 117-168.

Shintani, H & Polonský 1997, Precision and quantitation in capillary electrophoresis, Handbook of Capillary Electrophoresis Applications, Chapman & Hall, UK, pp. 41-56.

Pre-Lab:

- Notify the demonstrator and clean up the spill before further accidents occur.

2. (i) H2PO4- is the weak acid.

(ii) HPO42- is the conjugate base.

3. Isoelectric point of a protein