3. A researcher investigated the a set of conditions for a protein with an isoelectric point of 6.5 and also bind to subjected to four calcium. This protein was independent treatments: (i) pH 6.4, (ii) 10% glycerol, (iii) 10mM CaCl₂, (iv) 40% ammonium sulphate. This was followed by centrifugation and estimation of protein in supernatant. The results are depicted in the graph below:

   

   Which of the following treatments best represents the results shown in the graph?

   (1) a = ammonium sulphate, b = glycerol, c = pH 6.4, d = CaCl₂

   (2) a = CaCl₂, b = glycerol, c = ammonium sulphate, d = pH 6.4

   (3) a = pH 6.4, b = CaCl₂, c = ammonium sulphate, d = glycerol

   (4) a = CaCl₂, b = pH 6.4, c = glycerol, d = ammonium sulphate

Investigating Protein Stability Under Varying Conditions

Protein stability is crucial for its function, and researchers analyze how different conditions affect its solubility. In this experiment, a protein with an isoelectric point of 6.5 and calcium-binding ability was subjected to four different treatments:

1. pH 6.4

2. 10% Glycerol

3. 10mM CaCl₂

4. 40% Ammonium Sulfate

After treatment, the samples were centrifuged, and protein concentrations in the supernatant were measured. The data were then graphically represented.

Interpreting the Effects of Each Treatment

1. Effect of Ammonium Sulfate (a)

Ammonium sulfate precipitates proteins, significantly reducing solubility and lowering the amount of protein detected in the supernatant.

2. Effect of pH 6.4 (b)

Since the pH is close to the isoelectric point, solubility decreases, but not as drastically as with ammonium sulfate.

3. Effect of Glycerol (c)

Glycerol stabilizes proteins by preventing aggregation, leading to increased solubility.

4. Effect of Calcium Chloride (d)

Calcium binding influences protein solubility based on structural interactions, impacting its stability.

Correct Answer: 4 (a = CaCl₂, b = pH 6.4, c = glycerol, d = ammonium sulfate)

The solubility trends indicate that calcium chloride affects solubility, pH 6.4 causes moderate precipitation, glycerol enhances solubility, and ammonium sulfate leads to the most significant precipitation.