Protein-binding interactions and their effects MCQs With Answer

Introduction: Protein-binding interactions and their effects MCQs With Answer is a focused revision resource tailored for M. Pharm students studying Modern Bio-Analytical Techniques (MPA 202T). This quiz set explores how drugs and ligands interact with plasma proteins (e.g., albumin, alpha1-acid glycoprotein), experimental methods for quantifying binding (equilibrium dialysis, ultrafiltration, SPR, ITC), binding kinetics and thermodynamics, and the clinical consequences of altered protein binding on distribution, clearance, and drug–drug interactions. Questions emphasize analytical pitfalls, interpretation of binding constants, cooperativity, site specificity and assay design considerations. Use these MCQs to deepen conceptual understanding and prepare for advanced laboratory and clinical problem-solving.

Q1. Which analytical technique directly measures changes in refractive index near a sensor surface to study real-time protein–ligand binding kinetics?

• Isothermal titration calorimetry (ITC)
• Surface plasmon resonance (SPR)
• Equilibrium dialysis
• Ultrafiltration

Correct Answer: Surface plasmon resonance (SPR)

Q2. In equilibrium dialysis used to determine plasma protein binding, a major source of error is:

• Incorrect calculation of Hill coefficient
• Nonspecific adsorption of drug to the dialysis membrane
• Overestimation of enthalpy changes
• Interference from refractive index shifts

Correct Answer: Nonspecific adsorption of drug to the dialysis membrane

Q3. Which plasma protein primarily binds acidic drugs and is the most abundant carrier affecting drug distribution?

• Alpha1-acid glycoprotein (AAG)
• Transferrin
• Albumin
• Gamma globulin

Correct Answer: Albumin

Q4. A drug with very high plasma protein binding (≥ 99%) is most likely to exhibit which characteristic clinically when displaced by a second drug?

• No change in free concentration or pharmacologic effect
• Large transient increase in free fraction with potential toxicity
• Decreased clearance due to reduced free drug
• Immediate elimination by kidneys regardless of binding

Correct Answer: Large transient increase in free fraction with potential toxicity

Q5. Scatchard analysis is used to estimate binding parameters; a curved Scatchard plot suggests:

• Single class of identical independent binding sites
• Non-specific binding only
• Multiple classes of binding sites or cooperative binding
• Zero-order kinetics

Correct Answer: Multiple classes of binding sites or cooperative binding

Q6. Which parameter obtained from SPR or ITC describes the ratio of dissociation rate to association rate for a binary protein–ligand interaction?

• Association rate constant (ka)
• Equilibrium dissociation constant (KD)
• Enthalpy change (ΔH)
• Stoichiometry (n)

Correct Answer: Equilibrium dissociation constant (KD)

Q7. In ultrafiltration assays for unbound drug, which artifact can lead to underestimation of free fraction?

• Membrane protein leakage
• Nonspecific binding of drug to filter device
• Excessive ionic strength of buffer
• Use of radiolabeled ligand

Correct Answer: Nonspecific binding of drug to filter device

Q8. A Hill coefficient (nH) significantly greater than 1 indicates:

• Negative cooperativity among binding sites
• Positive cooperativity among binding sites
• Non-specific linear binding
• Single-site independent binding

Correct Answer: Positive cooperativity among binding sites

Q9. Which of the following best explains why changes in plasma protein binding often have a greater effect on total plasma concentration than on unbound concentration?

• Unbound concentration is tightly buffered by tissue binding
• Clearance mechanisms typically remove only bound drug
• Total concentration includes both bound and unbound fractions, so displacement alters total more dramatically while free concentration is regulated by redistribution and clearance
• Protein binding occurs only in tissues, not plasma

Correct Answer: Total concentration includes both bound and unbound fractions, so displacement alters total more dramatically while free concentration is regulated by redistribution and clearance

Q10. Alpha1-acid glycoprotein (AAG) preferentially binds which class of drugs, affecting their pharmacokinetics during acute-phase reactions?

• Acidic drugs
• Neutral hydrophobic drugs
• Basic (cationic) drugs
• Highly polar ionic drugs

Correct Answer: Basic (cationic) drugs

Q11. When performing ITC to analyze protein–ligand interaction thermodynamics, which output parameter directly indicates whether binding is enthalpy- or entropy-driven?

• Stoichiometry (n)
• Equilibrium constant (K)
• Change in Gibbs free energy (ΔG) and relative contributions of ΔH and –TΔS
• Association rate constant (ka)

Correct Answer: Change in Gibbs free energy (ΔG) and relative contributions of ΔH and –TΔS

Q12. Stereoselective protein binding can cause enantioselective pharmacokinetics. Which statement is true?

• Both enantiomers always bind identically to plasma proteins
• Differences in binding affinity of enantiomers can alter their unbound fractions and thereby their clearance and effect
• Protein binding does not affect enantiomeric disposition since binding is non-specific
• Enantioselective binding only occurs with albumin, not with AAG

Correct Answer: Differences in binding affinity of enantiomers can alter their unbound fractions and thereby their clearance and effect

Q13. Which experimental approach is most appropriate for measuring very tight binding (KD in low nM to pM range) between a drug and a plasma protein?

• Equilibrium dialysis with short dialysis times
• High-throughput ultrafiltration at room temperature
• Surface plasmon resonance (SPR) with kinetic fitting
• Simple protein precipitation followed by HPLC

Correct Answer: Surface plasmon resonance (SPR) with kinetic fitting

Q14. Which effect describes a scenario where decreased plasma albumin concentration increases free drug fraction but results in minimal change in pharmacological effect due to increased clearance of the free drug?

• Free-drug paradox
• Albumin trapping
• Compensatory clearance effect
• Hysteresis

Correct Answer: Compensatory clearance effect

Q15. In a competitive displacement interaction, a drug that displaces another from albumin will most likely cause which immediate change in the displaced drug?

• Decrease in free concentration
• Increase in free concentration
• Permanent decrease in total clearance
• Complete elimination of protein binding sites

Correct Answer: Increase in free concentration

Q16. During mass spectrometric measurement of free fraction, which pre-analytical consideration is critical to avoid artifactual underestimation of unbound drug?

• Heating the sample to denature protein
• Using plasticware with high nonspecific binding without validation
• Adding excess albumin to plasma before analysis
• Diluting sample with organic solvent to precipitate proteins

Correct Answer: Using plasticware with high nonspecific binding without validation

Q17. Allosteric modulators of albumin binding sites change ligand affinity by:

• Only increasing the number of binding sites
• Altering protein conformation and thereby changing the affinity at distant sites
• Destroying the ligand chemically
• Removing albumin from plasma

Correct Answer: Altering protein conformation and thereby changing the affinity at distant sites

Q18. According to the Van’t Hoff equation, an observed decrease in binding affinity with increasing temperature suggests that the binding process is primarily:

• Endothermic (ΔH > 0)
• Exothermic (ΔH < 0)
• Entropy-neutral
• Independent of ΔH

Correct Answer: Exothermic (ΔH < 0)

Q19. Which statement best distinguishes ultrafiltration from equilibrium dialysis for free drug measurement?

• Ultrafiltration achieves thermodynamic equilibrium across a membrane; equilibrium dialysis does not
• Equilibrium dialysis can be slower but minimizes pressure-induced dissociation, while ultrafiltration is faster but may disturb binding equilibrium and cause nonspecific adsorption
• Both methods are equally prone to membrane adsorption and give identical results in all cases
• Ultrafiltration requires radiolabeled drug while equilibrium dialysis does not

Correct Answer: Equilibrium dialysis can be slower but minimizes pressure-induced dissociation, while ultrafiltration is faster but may disturb binding equilibrium and cause nonspecific adsorption

Q20. Which clinical condition would most likely increase the unbound fraction of an acidic drug that is extensively albumin-bound?

• Dehydration with hemoconcentration
• Hypoalbuminemia due to liver failure
• Elevated AAG during acute inflammation
• Hyperbilirubinemia without protein changes

Correct Answer: Hypoalbuminemia due to liver failure

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