Amorphine-binding substance was isolated from brain tissue. The material was pur

Question

Amorphine-binding substance was isolated from brain tissue. The material was purified to homogeneity and identified as a glycoprotein of MW 260,000.  A solution of glycoprotein (030 mg/mL) was dialyzed against an equal volume of a solution of 3H-morphine.  At equilibrium, the chamber containing the glycoprotein contained

1.43 X 10-6 M total (bound + free) 3H-morphine.The chamber without the glycoprotein contained 0.78 X 10-6 M 3H-morphine.

Calculate (a) the concentration of bound3H-morphine, (b) the concentration of free protein, and (c) the dissociation constant for the glycoprotein-morphine complex. Assume one binding site per protein molecule.

>Please explain the steps to obtain the answer. Just throwing equations and answers will not help me learn.

Explanation / Answer

A morphine-binding substance was isolated from brain tissue. The material was purified to homogeneity and identified as a glycoprotein of MW 260,000.  A solution of glycoprotein (030 mg/mL) was dialyzed against an equal volume of a solution of 3H-morphine.  At equilibrium, the chamber containing the glycoprotein contained

1.43 X 10-6 M total (bound + free) 3H-morphine.The chamber without the glycoprotein contained 0.78 X 10-6 M 3H-morphine.

Calculate (a) the concentration of bound3H-morphine,

Total 3H-morphine Concentration(bound + free)=1.43 X 10-6 M

3H-morphine Concentration (free)=0.78 X 10-6 M

3H-morphine Concentration (bound)=total -free=1.43 X 10-6 M -0.78 X 10-6 M

=6.5 X 10-7 M but since equal volume of glycoprotein was added there original Conc wasdouble

3H-morphine Concentration (bound)=1.3 X 10-6 M

(b) the concentration of free protein,

3H-morphine Concentration (free)=1.56 X 10-6 M

and (c) the dissociation constant for the glycoprotein-morphine complex. Assume one binding site per protein molecule.

Dissciation Constant=Conc free/total concentration=(1.56 X 10-6 M)/(2.86 X 10-6 M)

=0.545

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