Question

Multi-part question according to lab textbook:

A student is analyzing crude cellular extract for protein content. Crude cellular extract is simply the cellular contents obtained by lysing (breaking open) cells. Which method (A280 or biuret) would be the most appropriate method for determining protein concentration and why?

A student has purified a blood protein with an extinction coefficient of 1.86 (mg cm/mL^-1) which the student compares to another blood protein which has an extinction coefficient of 0.72 (mg cm/mL^-1). Which protein has a greater number of aromatic residues per mg of protein? How did you make this determination?

The student measures the A280 of a sample of her purified blood protein (1.86 mg cm/mL^-1). The absorbance is 0.882. What is the concentration of her protein sample?

If the A280 and the biuret methods were in close agreement, what would this result suggest about the identity and purity of the unknown protein sample?

If the two methods (A280 and biuret) were not in close agreement, what does this suggest about the identity and purity of the unknown protein sample?

Answer 1

Multi-part question according to lab textbook:
A student is analyzing crude cellular extract for protein content. Crude cellular extract is simply the cellular contents obtained by lysing (breaking open) cells. Which method (A280 or biuret) would be the most appropriate method for determining protein concentration and why?
Answer: Now, biuret method is suitable for protein with high concentration while A280 method is more useful when protein concentrations are of lower range. Now, in cellular extract the protein concentration will not be very high which can react with biuret to give result. So, A280 method is more suitable.

A student has purified a blood protein with an extinction coefficient of 1.86 (mg cm/mL^-1) which the student compares to another blood protein which has an extinction coefficient of 0.72 (mg cm/mL^-1). Which protein has a greater number of aromatic residues per mg of protein? How did you make this determination?
Answer: The band at 280 nm for protein depends on the number of aromatic residues present in the protein or the extinction coefficient at 280 nm is the sum of all extinction co-efficient at 280 or of all the aromatic amino acids of the protein. Aromatic groups have absorption maxima in the range of 260-290 nm. So, the protein with extinction coefficient of 1.86 has greater number of aromatic residues.

The student measures the A280 of a sample of her purified blood protein (1.86 mg cm/mL^-1). The absorbance is 0.882. What is the concentration of her protein sample?
Answer: we know, A=εcl
0.882=1.86×c×1
concentration= 0.474 mg/mL

If the A280 and the biuret methods were in close agreement, what would this result suggest about the identity and purity of the unknown protein sample?

Answer: A280 gives idea about the number or aromatic amino acid residues of protein while biuret method is directly dependent on the peptide bonds present in the protein. If both of this experiments are supporting each other then the number of aromatic amino acid residue and peptide bonds are equivalent in number which tells that the protein is pure giving similar data in both.
If the two methods (A280 and biuret) were not in close agreement, what does this suggest about the identity and purity of the unknown protein sample?
Answer: If both are not giving same result then there is a high chance that the protein is not pure or a mixture of more than one protein which results in different result in A280 and biuret test. As two different Cu2+-complexes will form in biuret test and the extinction coefficient and absorbance will not match.