Question 1

The liquid emerging from the bottom of a chromatography column is called the .

Accepted Answer

The liquid emerging from the bottom of a chromatography column is called the eluate.

Question 2

Which is used as the basis of separation of proteins by affinity chromatography?

Accepted Answer

Affinity chromatography separates proteins based on the selective binding of the protein to a ligand on the column matrix. This ligand is chosen to specifically bind to the protein of interest, allowing for purification and isolation of the protein from other components in the mixture. The net charge, pI, molecular weight, and density of the protein are not directly used as the basis of separation in affinity chromatography.

Question 3

What is the net charge on the dipeptide Arg-Pro at pH 9.0? The table below gives the pKa?s of the ionizable groups on the free amino acids. $\begin{array}{|l|c|c|c|}
\hline \text { Free amino acid } & \begin{array}{c}
\text { pKa of } \
\alpha \text {-Carboxyl group }
\end{array} & \begin{array}{c}
\text { pKa of } \
\alpha \text {-amino group }
\end{array} & \begin{array}{c}
p \text { Ka of side } \
\text { chain }
\end{array} \
\hline \text { Arginine } & 1.8 & 9.0 & 12.5 \
\hline \text { Proline } & 2.0 & 10.6 & \cdots \
\hline
\end{array}$

Accepted Answer

The pKa of the α-amino group of Arg is 9.0, which means that at pH 9.0, half of the α-amino groups will be protonated and half will be unprotonated. The pKa of the side chain of Arg is 12.5, which means that at pH 9.0, almost all of the side chains will be protonated. The pKa of the α-carboxyl group of Pro is 2.0, which means that at pH 9.0, almost all of the α-carboxyl groups will be deprotonated. Therefore, the net charge on the dipeptide Arg-Pro at pH 9.0 will be +0.5.

Question 4

Which is used to hydrolyze the peptide bonds of a protein to yield free amino acids?

Accepted Answer

HCl (hydrochloric acid) is commonly used to hydrolyze the peptide bonds of proteins, breaking them down into free amino acids. This process typically involves the use of strong acids under high temperature.

Question 5

Which amino acids are linked in phenylalanylglycine?

Accepted Answer

Phenylalanylglycine is a dipeptide composed of the amino acids phenylalanine and glycine.

Question 6

In a biochemistry laboratory a student added ammonium sulfate to a tube containing a buffered protein solution. The student then centrifuged the solution. What was the student probably trying to do?

Accepted Answer

The student was likely trying to selectively precipitate and purify a certain protein. Ammonium sulfate precipitation is a common technique used to fractionate proteins based on their solubility.

Question 7

During the fractionation process which of the following remain in the supernatant?

Accepted Answer

During fractionation, the supernatant typically contains the target protein and other more soluble proteins, as they remain dissolved in the liquid phase after centrifugation.

Question 8

Which substance is used to fractionate proteins based on differences in their solubility as a function of salt concentration?

Accepted Answer

Ammonium sulfate is commonly used in a process called "salting out," which exploits differences in protein solubility at varying salt concentrations to fractionate proteins.

Question 9

The peptide bond is which of the following?

Accepted Answer

A peptide bond is an amide bond formed between the carboxyl group of one amino acid and the amino group of another.

Question 10

What is the net charge on the tripeptide Gly-Arg-Lys at pH 7? The table below gives the pK_a?s of the ionizable groups on the free amino acids. $\begin{array}{|l|c|c|c|} \hline \text { Free amino acid } & \begin{array}{c} \mathrm{pKa} \text { of } \ \alpha \text {-Carboxyl group } \end{array} & \begin{array}{c} p \mathrm{k} \text { of } \ \alpha \text {-amino group } \end{array} & \begin{array}{c} \mathrm{pK} \mathrm{a} \text { of side } \ \text { chain } \end{array} \ \hline \text { Glycine } & 2.4 & 9.8 & \cdots \ \hline \text { Arginine } & 1.8 & 9.0 & 12.5 \ \hline \text { Lysine } & 2.2 & 9.1 & 10.5 \ \hline \end{array}$

Accepted Answer

At pH 7, the carboxyl groups are deprotonated (-1 each), and the amino groups are protonated (+1 each). Glycine contributes 0 net charge. Arginine's side chain is protonated (+1), and Lysine's side chain is protonated (+1). Total net charge: +2.

Question 11

What is the purpose of SDS in SDS-PAGE?

Accepted Answer

SDS (sodium dodecyl sulfate) is a detergent that denatures proteins and imparts a uniform negative charge, allowing them to be separated based on molecular weight during electrophoresis.

Question 12

In which technique is a protein solution a pumped through a metal needle at high voltage to create tiny droplets that are analyzed for their mass/charge ratio?

Accepted Answer

Electrospray mass spectrometry involves pumping a protein solution through a metal needle at high voltage to create tiny charged droplets, which are then analyzed for their mass/charge ratio.

Question 13

Which technique is used less for protein purification and more for the determination of molecular weights?

Accepted Answer

SDS-PAGE is a technique used for the separation of proteins based on their molecular weight. It is commonly used for the determination of molecular weights of proteins, as well as for the analysis of protein complexes and protein-protein interactions.

Question 14

What is the N-terminal for the pentapeptide Val-Ile-Glu-Arg-Tyr?

Accepted Answer

The N-terminal of a peptide is the amino group of the first amino acid. In this case, the first amino acid is valine, so the N-terminal is the NH3+ group on the side chain of valine.

Question 15

Even though mass spectrometry has been in use for over a hundred years, it had only limited use with proteins until the 1980ʹs because

Accepted Answer

Mass spectrometry's limited use with proteins until the 1980s was primarily due to the difficulty in ionizing and vaporizing proteins without decomposition, making it challenging to disperse charged proteins into a gaseous stream of particles for analysis.

Question 16

Which technique is most sensitive and accurate for the determination of a proteinʹs molecular weight?

Accepted Answer

Mass spectrometry is the most sensitive and accurate technique for determining a protein's molecular weight, as it can measure the mass-to-charge ratio of ions with high precision.

Question 17

The ionic charges associated with a protein molecule are .

Accepted Answer

The ionic charges associated with a protein molecule are mostly contributed by the side chains of constituent amino acids. The α-carboxyl group and α-amino group contribute to the net charge of the protein, but this contribution is small compared to that of the side chains. The N-terminal and C-terminal residues may contribute to the ionic charges but are not the only contributors. The ionic charges are also pH-dependent, but they are not independent of the amino acid composition.

Question 18

A mixture of four proteins X, Y, Z and N is applied to a gel-filtration column. Given the information supplied, which will elute first? $\begin{array}{|c|c|}
\hline \text { Protein } & \text { Molecular Weight g/mol) } \
\hline X & 35,000 \
\hline Y & 26,000 \
\hline Z & 146,000 \
\hline \mathrm{N} & 26,000 \
\hline
\end{array}$

Accepted Answer

In gel-filtration chromatography, larger molecules elute first because they are less likely to enter the pores of the gel and thus travel a shorter path through the column. Protein Z, with the highest molecular weight of 146,000 g/mol, will elute first.

Question 19

Gel-filtration chromatography separates a mixture of proteins on the basis of .

Accepted Answer

Gel-filtration chromatography separates proteins based on their size. Proteins of different sizes travel through the column matrix at different rates and are thus separated from each other. The larger proteins pass through the column more quickly and are eluted earlier, while smaller ones are retained longer and elute later. The other options, charge, affinity for ligands in the column matrix, and density, are not factors that affect separation in gel-filtration chromatography.

Question 20

A mixture of two proteins with the same pI and molecular weights MW) of 10,000 and 15,000 daltons, respectively, are applied to a gel filtration column. What happens during elution?

Accepted Answer

Gel filtration chromatography separates molecules based on size, with larger molecules eluting first because they cannot enter the pores of the gel matrix as easily as smaller molecules.

Quiz 3: Amino Acids and the Primary Structures of Proteins