Enzyme Technology Questions and Answers – Enzymes Purification – Affinity Chromatography

This set of Enzyme Technology Multiple Choice Questions & Answers (MCQs) focuses on “Enzymes Purification – Affinity Chromatography”.

1. The chromatographic method of separating biochemical mixture of compounds, based on highly specific biological interactions is referred to as ______________
a) thin layer chromatography
b) ion-exchange chromatography
c) affinity chromatography
d) gel permeation chromatography
View Answer

Answer: c
Explanation: Affinity chromatography is a method based on separation of molecules based on specific biological interaction such as antigen-antibody, enzyme-substrate etc. Thin layer chromatography is a technique which separates molecule based on their solubility in stationary or mobile phase. Ion-exchange chromatography is a technique based on separation of molecules using the ions present in them. Gel permeation chromatography is based on separation of molecules based on their size.
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2. Which of the following is not a highly specific biological interaction to be used in affinity chromatography?
a) Cations-anions
b) Antigen-antibody
c) Enzyme-substrate
d) Receptor-ligand
View Answer

Answer: a
Explanation: Cations-anions are used in ion-exchange chromatography to separate molecules based on the ions present in them. Antigen-antibody, enzyme-substrate, receptor-ligand are used in affinity chromatography to separate highly specific biological interactions molecules.

3. Which of this is not true while selecting a solid matrix or column during affinity chromatography?
a) The matrix should interact weakly with enzymes
b) The matrix should be based on inorganic compounds
c) The matrix should exhibit good flow property
d) The matrix should be highly porous
View Answer

Answer: b
Explanation: The matrix should be based on inorganic compounds is true in case of ion-exchange chromatography. The following are true in case of matrix selection during affinity chromatography.
• The matrix should interact weakly with enzymes in order to minimize non-specific adsorption of other enzymes.
• The matrix should exhibit good flow property.
• The matrix should be highly porous when there is relatively weak affinity between the ligand and protein.
• The matrix should be chemically and mechanically stable.
• The matrix must possess certain chemical groups.

4. In affinity chromatography, the matrix should be chemically and mechanically stable.
a) True
b) False
View Answer

Answer: a
Explanation: In affinity chromatography, the matrix selected should be chemically and mechanically stable because of coupling conditions and varying conditions pH, ionic strength, temperature and presence of denaturants which may be needed for adsorption or elution. Due to this, it permits the repeated use of certain specific adsorbents. Hence the above statement is true.

5. The matrix selected during affinity chromatography must possess certain chemical groups.
a) True
b) False
View Answer

Answer: a
Explanation: In affinity chromatography, the matrix must possess certain chemical groups which can be activated or modified in order to allow the chemical linkage of variety of ligands under conditions which are harmful to the matrix. Hence the above statement is true.
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6. The first step in preparation of affinity chromatography column is _____________
a) ligand attachment to matrix
b) coupling of aromatic amines to matrix
c) activation process
d) precipitation
View Answer

Answer: c
Explanation: Activation process is the first step in preparation in affinity chromatography column which involves introduction of reactive groups into the chemically inert polymeric matrix material. The second step is attaching the ligand to the matrix covalently. After the activation, the activated matrix may be coupled to primary aliphatic or aromatic amines. Elution is the process of removing the adsorbed material from the column. This is the last step in affinity chromatography.

7. __________ refers to eluting a compound by creating a competition between the bounded enzymes with its suitable complimentary protein in solution.
a) Specific elution
b) Nonspecific elution
c) Filtration
d) Dialysis
View Answer

Answer: a
Explanation: In affinity chromatography, when the elution is done by creating a competition between the bounded enzymes with its suitable complementary protein in solution, the elution is referred to as specific elution. Nonspecific elution is done when specific ligand fail to elute desired enzyme. Filtration is separation method based on the size. Dialysis is a separation technique in which large molecules are retained in the membrane eluting the smaller molecules.

8. Which among these is not a way of non-specific elution?
a) Solvent change
b) pH change
c) Reversible denaturation
d) Competition to suitable complementary protein
View Answer

Answer: d
Explanation: Nonspecific elution can be done by using solvent or buffer changes, pH or ionic strength changes, the application of chaotropic reagents, temperature changes, reversible denaturation and destruction of spacer arm. Competition to suitable complementary protein is a type of specific elution.

9. Acetylcholine esterase can be purified by using affinity chromatography with the use of _______ as a ligand.
a) ATP
b) NADP
c) Acrdinium
d) Con A
View Answer

Answer: c
Explanation: For acetylcholine esterase, acrdinium can be used as ligand. ATP can be used as ligand to purify adenosine kinase. Alcohol dehydrogenase is purified by using NADP as a ligand. Con A and mannose is used to purify acid phosphatase.
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10. Acid phosphatase: Mannose:: Alpha amylase: ______________
a) KCl
b) Glycogen
c) Adenosine
d) Methyl-D-mannoside
View Answer

Answer: b
Explanation: For purifying acid phosphatase, the eluent used is Mannose. In the same way, alpha amylase can be purified by using glycogen as an eluent. Adenosine is used as an eluent for purifying adenosine kinase and methyl-D-mannoside is used as an eluent for acetylcholine esterase.

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Manish Bhojasia, a technology veteran with 20+ years @ Cisco & Wipro, is Founder and CTO at Sanfoundry. He is Linux Kernel Developer & SAN Architect and is passionate about competency developments in these areas. He lives in Bangalore and delivers focused training sessions to IT professionals in Linux Kernel, Linux Debugging, Linux Device Drivers, Linux Networking, Linux Storage, Advanced C Programming, SAN Storage Technologies, SCSI Internals & Storage Protocols such as iSCSI & Fiber Channel. Stay connected with him @ LinkedIn | Youtube | Instagram | Facebook | Twitter