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
1.2 Proteins different in size can be separated by gel-filtration(size-exclusion, molecular sieve) chromatography. 1. 2.1 Samples are applied to columns of porous beads(made of insoluble but highly hydrated polymers like dextran, agarose, and polyarylamide)
1.2 Proteins different in size can be separated by gel-filtration (size-exclusion, molecular sieve) chromatography. 1.2.1 Samples are applied to columns of porous beads (made of insoluble but highly hydrated polymers like dextran, agarose, and polyarylamide)
The stationary phase is composed of a porous matrix and absorbed immobile solvent The mobile phase is the flowing solvent consisted of buffers and salts (fig
The stationary phase is composed of a porous matrix and absorbed immobile solvent. The mobile phase is the flowing solvent consisted of buffers and salts. (fig.)
rapidly through the column and emerge irs 1. 2. 2 Larger protein molecules flow mor because the ley cannot enter the internal volume of the beads 1.2.3 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.) 1.2.4 Gel-filtration usually has low resolution
1.2.2 Larger protein molecules flow more rapidly through the column and emerge first because they cannot enter the internal volume of the beads. 1.2.3 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.) 1.2.4 Gel-filtration usually has low resolution
1. 3 The solubility of most proteins is lowered at high salt concentrations 1.3.1 This effect is called salting out 1.3.2 The dependence of solubility on salt concentration differs from one protein to another, hence salting out can be used to fractionate proteins. 1.3.3 Ammonium sulfate precipitatio often used in fractionating proteins(also to concentrate proteins). 1.3.4 The mechanism of salting out is not well understood. Dehydration is a possible cause)
1.3 The solubility of most proteins is lowered at high salt concentrations. 1.3.1 This effect is called salting out. 1.3.2 The dependence of solubility on salt concentration differs from one protein to another, hence salting out can be used to fractionate proteins. 1.3.3 Ammonium sulfate precipitation is often used in fractionating proteins (also to concentrate proteins). 1.3.4 The mechanism of salting out is not well understood. (Dehydration is a possible cause)
1. 4 Proteins can be separated on the basis of their net charge by ion exchange chromatography 1.4.1 Proteins have different net charge at a given ph due to their differences in pl values (as for amino acids). 1. 4.2 Proteins bind to charged resins with difierent affinity, thus being able to be released (eluted)at different salt concentrations or ph values(using buffers with a gradient of salt or ph is run through the column)
1.4 Proteins can be separated on the basis of their net charge by ion exchange chromatography 1.4.1 Proteins have different net charge at a given pH due to their differences in pI values (as for amino acids). 1.4.2 Proteins bind to charged resins with different affinity, thus being able to be released (eluted) at different salt concentrations or pH values (using buffers with a gradient of salt or pH is run through the column)