Which method is used to separate bound T4 from its carrier?

• A. Equilibrium dialysis
• B. Ultrafiltration
• C. Gel filtration
• D. Spectrophotometry

Answer: (A)

Separating bound from free hormone relies on using a barrier that only allows the small, unbound molecules to pass while keeping the larger protein-bound complexes on the original side. In equilibrium dialysis, the serum sample is placed on one side of a semipermeable membrane and a buffer on the other. Free T4, being a small molecule, diffuses across the membrane until the concentration of free T4 is the same on both sides. The T4 that remains attached to its carrier protein stays with the high-molecular-weight complex on the serum side and does not cross the membrane. Once equilibrium is reached, measuring the T4 in the dialysate reflects the free fraction, which is precisely what you want when determining how much T4 is available to tissues.

This method is preferred for the free T4 assessment because it preserves the natural binding equilibrium and directly isolates the unbound portion without disrupting the interaction between T4 and its carrier. Other methods, like ultrafiltration, can separate based on size but may alter binding dynamics or be affected by membrane interactions and processing conditions, making them less ideal as the reference approach. Gel filtration separates by size but is slower and less practical for routine free hormone measurements, and spectrophotometry describes detection rather than separation.