Chapter 7 Techniques for Exploring proteins
Chapter 7 Techniques for Exploring Proteins
Assay:化验,分析,测定。 Enzyme activity: l unit (U)is defined as the amount of enzyme which convert 1 micromole (umol) of substrate to product(s)in l minute under specified conditions Specific activity: enzyme activity expressed per unit mass of protein present.(units/mg)
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
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
ble 5-5 A Purification Table for a Hypothetical Enzyme* Fraction Total Procedure volume protein Specific activity or step (m) (mg) (units) (units/mg) 1. Crude cellular extract 1,40010,000100,000 10 2. Precipitation with ammonium sulfate 280 3,000 96,000 32 3. lon-exchange chromatography 0 400 80.000 200 4. Size-exclusion chromatography 100 60.000 600 5. Affinity chromatog raphy 6 45,000 15000 " All data represent the status of the sample after the designated procedure has been carried out. Activity and specific activity are defined on page 137
1. Proteins in native conformation can be purified (separated) according to their size, solubility, charge, and binding aflinity 1.1 Proteins can be separated from small molecules by dialysis thanon a semipermeable membranes 1.1.1 Molecules significantly larger than the diameter of the membrane pores are retained in the dialysis bag, whereas small ones diffuse out
1. Proteins in native conformation can be purified (separated) according to their size, solubility, charge, and binding affinity. 1.1 Proteins can be separated from small molecules by dialysis through a semipermeable membranes. 1.1.1 Molecules significantly larger than the diameter of the membrane pores are retained in the dialysis bag, whereas small ones diffuse out