techniques for exploring proteins(Ch. 7).ppt

Chapter 7 Techniques for Exploring Proteins
Assay: 化验,分析,测定。
Enzyme activity: 1 unit (U) is defined as the amount of enzyme which convert 1 micromole (mmol) of substrate to product(s) in 1 minute under specified conditions.
Specific activity: enzyme activity expressed per unit mass of protein present. (units/mg)
Red balls=active enzymes. Other color balls=other proteins. The total activity is the same for the two cups but the specific activity is higher for the right cup.
1. Proteins in native conformation can be purified (separated) according to their size, solubility, charge, and binding affinity.
Proteins can be separated from small molecules by dialysis through a semipermeable membranes.
Molecules significantly larger than the diameter of the membrane pores are retained in the dialysis bag, whereas small ones diffuse out.
Proteins different in size can be separated by gel-filtration (size-exclusion, molecular sieve) chromatography.
Samples are applied to columns of porous beads (made of insoluble but highly hydrated polymers like dextran, agarose, and polyarylamide)
The stationary phase posed of a porous matrix and absorbed immobile solvent.
The mobile phase is the flowing solvent consisted of buffers and salts. (fig.)
Larger protein molecules flow more rapidly through the column and emerge first because they cannot enter the internal volume of the beads.
Proteins smaller than the diameter of the pores on the beads will enter the labyrinthian path of the beads, and hence, are slowed in mobility. (Manufacturer controls the properties, such as pore size distribution, of the beads.)
Gel-filtration usually has low resolution.
The solubility of most proteins is lowered at high salt concentrations.
This effect is called salting out.
The dependence of solubility on salt concentration differs from one protein to another, hence salting out can be used to fractionate proteins.
Ammonium sulfate prec