Enzyme Characterization - Biochemistry - Lecture Slides, Slides of Biochemistry

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Module 4:

Enzyme purification

Module 4:

Enzyme purification and characterization

Lecture5: Enzyme purification

and characterizationand characterization

Protein Purification

•^ In an organism any one protein is present as a very•^ In an organism, any one protein is present as a verysmall percentage of the total biomolecules.• In order to characterize a protein fully, it isIn order to characterize a protein fully, it isnecessary to purify it.• There are many possible strategies that one coulduse to purify a protein.• In general, the more that you know about thet i '^

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protein's properties, the better strategy you couldadopt to purify it.• A typical protein purification strategy will beA typical protein purification strategy will becomprised of several stages, each one takingadvantage of different characteristics of the protein.g

p

Modulating Solubility

1) Precipitation at the pI A protein's average net charge at its isoelectricpoint is 0. Above or below this pH, the proteinmolecules are negatively or positively charged,respectively, causing them to repel each other.

Modulating Solubility

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2) Salting in)^

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Many proteins are poorly soluble in pure water,but are much more soluble in salt-containing

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solutions. Thus, lowering ionic strength can beused to precipitate certain proteins.

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Ammonium sulfate precipitation

• Precipitation with ammonium sulfate is acommon first step in protein purification.• Because proteins precipitate at differentconcentrations of salt, one can perform afirst "cut" with a concentration that willleave the desired protein soluble, removethe precipitate, and then add sufficient saltto precipitate the desired protein.

Ammonium sulfate precipitationExample:Example: • Your protein will remain soluble at 30% (w/v) ammoniumsulfate, but precipitates at 40% ammonium sulfate.• You slowly add the salt to your protein extract until itreaches a concentration of 30%, allow precipitation to

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occur, and then centrifuge the solution to remove theprecipitate.• You take the supernatant, and add ammonium sulfate untilit reaches a concentration of 40%, allow precipitation tod th

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occur, and then centrifuge the solution to remove theprecipitate.• You dissolve the precipitate in a buffer and dialyze it toremove the salt.

Dialysis

Gel filtration chromatography

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(a.k.a. "molecular sieve", "size exclusion")• The protein is applied to the top of a column consisting ofporous beads made of a hydrated material, such as agarose,dextran, or polyacrylamide.,^

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• The pores of the beads are of a controlled size and thisregulates which proteins can enter the beads.• The larger proteins will be excluded from the beads and will• The larger proteins will be excluded from the beads and willflow through the column faster than the smaller molecules,which experience a much larger volume.i

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• In practice, there exists a range of pore sizes, and proteins areseparated by their sizes (and shapes): the largest are elutedfirst, and the smallest elute last.,

Gel filtration chromatography

• V^ = Vbed^

• V^ beads^ voidbed beads^ void

• The^ void volume

is the volume surrounding the beads.

• The^ bed volume

is the total volume of the column.

• V^ is typically about Vid^

/3b d V^ is typically about Vvoid^

/3bed^

• A protein can be characterized by its elution volume (Velution),hi h i^ th

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f^ l^ t^

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which is the volume of solvent required to elute it from thecolumn. The relative elution volume (=V

/ V^ elution void ) is a quantity

independent of the particular column used.• By standardizing the column with proteins of known size, onecan use this technique to estimate molecular weight (assuming thatcan use this technique to estimate molecular weight (assuming thatthe shape is close to that of the standards – more or less globular).

Commonly Used Gel Filtration Materials

Ion exchange chromatography

• In most cases, a column is prepared with the ion exchanger,which is then equilibrated with the same buffer used towhich is then equilibrated with the same buffer used todissolve the protein. The exchanger and pH are chosen suchthat the protein will bind relatively tightly to the ion exchanger.• The protein solution is loaded to the top and the column iswashed with the buffer, removing all proteins with the

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opposite charge or low charge.• The protein can be eluted from the column either by changing• The protein can be eluted from the column either by changingthe pH or by increasing the salt concentration, which shieldsthe charges and thus decreases their attraction. Elution is mostf^

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often carried out by applying a salt gradient to the column.

Device for generating a linear concentration gradient.

Molecular formulas of cellulose based ion exchangersMolecular

formulas of cellulose based ion exchangers.