Purification and characterization of human transcortin

Area of Science:

• Biochemistry
• Protein Chemistry

Background:

• Human transcortin is a key plasma protein responsible for binding and transporting cortisol.
• Understanding transcortin's properties is crucial for studying steroid hormone regulation.

Purpose of the Study:

• To purify human transcortin to homogeneity.
• To characterize its cortisol-binding activity and heterogeneity.

Main Methods:

• Two-step purification: affinity chromatography with cortisol hemisuccinate and hydroxyapatite chromatography.
• Sodium dodecyl sulfate/polyacrylamide-gel electrophoresis (SDS-PAGE) for purity assessment.
• Equilibrium dialysis for determining cortisol-binding affinity.
• Isoelectric focusing (IEF) to analyze protein heterogeneity.

Main Results:

• Purified transcortin showed a single band on SDS-PAGE, indicating homogeneity.
• High apparent association constant (2.5 X 10^8 M^-1) for cortisol binding at 4°C.
• Isoelectric focusing revealed six distinct bands for native transcortin, with a possible desialylated form at pH 6.15.
• Desialylated transcortin also showed six bands on IEF, with pI values ranging from 4.90 to 6.30.

Conclusions:

• Human transcortin can be effectively purified using a combination of affinity and hydroxyapatite chromatography.
• The purified transcortin exhibits significant cortisol-binding capacity.
• Isoelectric focusing demonstrates heterogeneity in purified transcortin, suggesting potential post-translational modifications like sialylation.