This document requires Netscape 3.x or compatible Web Browser.

---

The University of Texas-Houston Health Science Center

Proteins - Analysis of Structure and Function

Lesson 2.0

Isolation - The Second Step

Instructions
Please enter both your name & social security number first below to be sure you get credit (before doing anything else). Then at your own pace. When Practice Exercises appear, click the appropriate button to choose your answer. Then press the "Get Feedback..." button to find out how you did. Continue to try again if you miss. After studying Lesson 2.0, and responding to all practice exercises, follow instructions at the end to submit your responses for Lesson 2.0 credit.
NAME:
Social Security # (No dashes please):

Lesson 2:Isolation - The Second Step 2.0 - 1

---

Proteins - Analysis of Structure and Function

Lesson 2: Isolation: The Second Step

Contents:

1. Fractional Precipitation
2. Centrifugation and Ultracentrifugation
3. Dialysis and Ultrafiltration
4. Chromatography
5. Electrophoresis

Lesson 2.0 Objectives:

Upon completing Lesson 2 and the practice exercises, the student should be able to:

1. Identify appropriate reagents for the fractional precipitation of proteins
2. Select a method for obtaining a crude isolate of protein from the extraction material
3. Identify the uses of centrifugation and ultracentrifugation in protein isolation
4. Relate the Svedburg unit to the sedimentation coefficient of a protein
5. Calculate the molecular weight of a protein from sedimentation coefficient data
6. Identify the basis of separation of molecules using dialysis
7. Justify the use of ultrafiltration rather than dialysis in protein purification
8. Identify a practical use for batch chromatography in protein purification
9. Justify the use of one type of chromatography over another in the determination of charge adsorption characteristics, solubility characteristics, molecular size and shape, affinity, and        isoelectric point
10. Calculate the yield and purification achieved following several chromatographic steps.
11. Identify the factors that influence electrophoretic mobility.
12. Calculate the electrophoretic mobility of a protein
13. Identify a practical use for electrophoresis in protein purification
14. Justify the use of one type of electrophoresis over another in the determination of isoelectric point, determination of purity, and separation of similar proteins.

Isolation or Purification

After the proteins have been extracted from the tissue or source, one must determine if the protein of interest is present in the extract. If the protein has a function its presence in the extract may be measured by its functional activity or unique characteristic. When the protein has been detected in the crude extract, the second step is to isolate it from other substances and other proteins. It is necessary to obtain the protein of interest in a pure and native form. It is also important to isolate the protein of interest in sufficient quantity to analyze its purity. This step may also be called "purification".

Isolation-How Do We Start?

The isolation of our protein begins by first performing cruder methods and following them by more refined methods of separation of this protein from other molecules.

These methods in some order of refinement are:

1. Fractional Precipitation
2. Centrifugation and Ultracentrifugation
3. Dialysis and Ultrafiltration
4. Chromatography
5. Electrophoresis

1.Fractional Precipitation

Learning Objectives:
Upon completing this topic and the practice exercises, the student should be able to:
1. Identify appropriate reagents for the fractional precipitation of proteins
2. Select a method for obtaining a crude isolate from the extraction material.

The separation of proteins from other compounds for years has begun by precipitation of the protein to be studied. The solubility of proteins in salt solutions has been used extensively to isolate crude preparations of proteins. In some concentrations, the addition of salt with improve the solubility of protein and other concentrations or in cases of other proteins, the addition of salt causes the protein to precipitate.

If the protein is known to be precipitated by a certain concentration of a salt solution, this may be used to obtain sizable amounts of a crude isolate. For example, albumin can be separated from globulins in human serum by precipitation of the globulins with a warm solution of sodium sulfate and sulfite with a total concentration of 27%. Myoglobin also may be separated from other muscle proteins by adding a 65% saturated solution of ammonium sulfate. Most other proteins are precipitated whereas the myoglobin remains in solution.In another case, Hemoglobin S (sickle cell hemoglobin), when deoxygenated with sodium hydrosulfite, forms a fine precipitate with potassium phosphate buffer (2.3mol/L). This solubility was the basis for a test for sickle cell disease for many years.

Fractional precipitation has also been done using varying concentrations of alcohol. Blood clotting factors were originally isolated this way. Organic acids such as trichloroacetic acid and sulfosalicylic acid will precipitate most proteins but they are not useful in the fractional isolation of specific proteins.

If one is working with a protein which has not been characterized but which has a function which can be assayed, then a titration of the solubility of the protein may be made. This is done by adding varying concentrations of a salt solution and measuring the function of the protein which remains in solution. It is useful to perform this titration on a small sample and use the results to begin initial isolation of large amounts of the protein to be studied.

Another method for preparing a crude isolate of the protein of interest is to perform batch chromatography.

Fractional Precipitation
Practice Exercise 2.1:	Which of the following is true regarding fractional precipitation? No Response Is best done by organic acids Is a way to prepare a crude isolate Ammonium salts can not be used Can not be used on small volumes

Lesson 2:Isolation - The Second Step 2.0 - 2

---

Proteins - Analysis of Structure and Function

2. Centrifugation and Ultracentrifugation

Learning Objectives:
Upon completing this topic and the practice exercises, the student should be able to:
1. Identify the uses of centrifugation and ultracentrifugation in protein isolation
2. Relate the Svedburg unit to the sedimentation coefficient of a protein
3. Calculate the molecular weight of a protein from sedimentation coefficient data

Removing Large Particles

Centrifugation is a method which allows for the separation of macromolecules according to the mass. Sedimentation in a rotor with fixed-angle tubes for 10 to 20 minutes at 2,000 to 4,000 rpm will usually separate soluble protein molecules from any particulate matter that remains in the extract.

Ultracentrifugation - Separation of Particles by Mass Shape and Density

The study of proteins using ultracentrifugation allows for separation of proteins and determination of their molecular weights in a non-denaturing environment.

Features other than the mass of the protein molecule also play a role in its centrifugation. These features are shape and density of the molecule. A particle with greater mass is propelled by a large centrifugal force to the bottom of the tube faster than one of lesser mass when they have equal shapes and density. Globular proteins will sediment faster than elongated or fibrous proteins with the same mass.

Under centrifugal force molecules in solution move away from the axis of rotation and give a sharp boundary between the solution and the pure solvent. The rate of movement of this boundary is the basis for the sedimentation velocity method of determining the sedimentation coefficient. The rate of movement is followed by photographing the boundary position detected by a Schlieren lens at timed intervals.

The boundary positions during ultracentrifugation of serum albumin (a) and gamma globulins (b) are shown below.

Sedimentation Coefficient

The sedimentation coefficient ( s) can be calculated from the rate of the movement of the boundary according to equation 2.1

s= (dc/dt) (1/w²r)    Equation 2.1

where (c) is the distance traveled by the protein in the time period, t, and (w²) is the angular velocity of the rotor in radians per second squared and r is the radius (cm) of the rotor. The number of radians per second is obtained by multiplying the speed of the centrifuge in revolutions per second (RPS) by two pi. The sedimentation coefficient is determined from three or more ultracentrifugation studies made on three or more differing concentrations of the proteins. The s_o is obtained by extrapolating the values for the sedimentation coefficient to zero concentration.

The sedimentation coefficient, s, is in units of seconds and that of protein molecules varies widely but usually is in the range between 1^-13 and 200^-13. The Svedburg unit, S , is expressed as the integer which would preceed the l0^-13.

Calculation of Sedimentation Coefficient/Svedburg Constant
Practice Exercise 2.2	Given the following data, select the correct value for the Svedburg Constant,S. The sedimentation velocity measurements were made at 20oC. and at 56,000 revolutions per minute in an ultracentrifuge at a radius of 8cm. Photographs were made at 16 minute intervals. cm traveled time(minutes) 0.0010 x 10-4 3 0.1873 x 10-4 19 0.3736 x 10-4 35 0.5599 x 10-4 51 No Response 70 7 3.3 0.7

Molecular Weight Calculation

The molecular weight can be calculated using the sedimentation coefficient using the formula below.

M = RTs / D(1-v^~p)  Equation 2.2

where R is the gas constant in ergs/mole per degree, T is absolute temperature, D is the diffusion coefficient and p is the partical specific volume. Examples of the S values and corresponding molecular weights are the molecular weight of serum albumin of 69,000 with an S of 4.6, and a molecular weight of a monoclonal immunoglobulin G was determined to be 178,000 and its S was determined as 7.08. While there is some relationship of S to the molecular weight, it is not directly linear because of shape and density considerations.

Calculation of Molecular Weight
Practice Exercise 2.3	Given the data below select the correct molecular weight: This protein has been analyzed by ultracentrifugation at 20o.The average sedimentation coefficient was 4.59 x 10-13. The average diffusion coefficient was 0.596 x 10 cm2/sec.The protein's partial specific volume was 0.736. Solutions of the protein at concentations between 0.39 and 0.57% were used. The density of water at 20oC is 0.998. 70,668 50,650 75,000 80,850 101,000

Lesson 2:Isolation - The Second Step 2.0 - 3

---

Proteins - Analysis of Structure and Function

3. Dialysis and Ultrafiltration

Learning Objectives:
Upon completing this topic and the practice exercises, the student should be able to:
1. Identify a practical use for dialysis in protein purification
2. Justify the use of ultrafiltration rather than dialysis in protein purification

Dialysis and ultrafiltration are useful in desalting the protein extract. In order to perform chromatographic separation of the protein of interest, it is frequently necessary to have a sample of low ionic strength.

The separation of large molecules from small ones is possible using membranes which have appropriate pore sizes. Membranes made of collodion or cellophane are available to use in dialysis. The membrane's pore size is selected to allow small molecules to pass through from the sample while the larger protein molecule is retained within the membrane. These membranes come in tubes in which the sample can be placed, the tube tied and placed in a beaker of buffer or water for dialysis to occur. The membranes also come in flat sheets that can be placed in holders which can be tightly placed between vessels holding the two solutions. Dialysis generally requires 12-24 hours.

Common Use for Dialysis
Practice Exercise 2.4	What is a common use of dialysis in purification of protein from an extract? No Response To separate proteins based on charge To denature unwanted proteins To rid the protein solution of large concentrations of salt To increase the volume of extract

Ultrafiltration is performed using similar membranes. Generally these are cellulose or cellulose acetate. Some type of force is usually applied to speed up the filtration process. In one case the membrane is placed in a holder which fits into a centrifuge tube. The sample is placed on top of the membrane in the centrifuge tube. Centrifugation causes the liquid and small solutes to pass through to the bottom of the tube while the protein is left on the membrane. Another method is to place the membrane over a vessel to which a vacuum is applied to cause the liquid and small solutes to pass through the membrane. These methods find use only in certain situations.

Dialysis Versus Ultrafiltration
Practice Exercise 2.5	What may justify the use of ultrafiltration rather than dialysis? No Response Ultrafiltration is faster Ultrafiltration results in better separation Dialysis requires special equipment Dialysis can not be used on small volumes

Lesson 2:Isolation - The Second Step 2.0 - 4

---

Proteins - Analysis of Structure and Function

4.Chromatography

Learning Objectives:
Upon completing this topic and the practice exercises, the student should be able to:
1. Identify a practical use for batch chromatography in protein purification.
2. Justify the use of one type of chromatography over another in the determination of charge, adsorption characteristics, solubility characteristics, molecular size and shape, affinity, and isoelectric point.
3. Calculate the yield and purification achieved following several chromatographic steps.

Chromatography is a term applied to the separation of compounds based on their size, shape, ionic charges, isoelectric points and special affinities for other molecules. Proteins and other molecules are separated by their partition between a stationary phase or solid support and a mobile phase. The mobile phase may be a liquid or gas while the stationary phase is usually a solid or a liquid attached most often to a solid support. Chromatography is done in glass (or metal) cylinders called columns, in flasks ("batch method"), or on slabs of cellulose or starch gel ("slab or layer chromatography").

A diagram below of a chromatograph is from "Introduction to High Performance Chromatography", a computer-based training program from SAVANT Audiovisuals Copyright 1991, 1998. Used with permission. The SAVANT program is recommended for those students who may require a complete introduction to high performance liquid chromatography(HPLC).

The protein is applied to the chromatographic medium, binds in some way, moves with mobile phase and eventually is eluted by the mobile phase. The methods described below in general terms are those most frequently employed in protein isolation. The fraction of the eluant containing the protein of interest is detected and collected. Chromatography methods are generally classified as:

A. Ion Exchange
B. Adsorption
C. Partition
D. Steric Exclusion
E. Affinity
F. Chromatofocusing

A. Ion Exchange Chromatography

This type of chromatography may be performed either by a batch method or using a column for the ion exchange medium,e.g.Diethylaminoethyl(DEAE)Sephadex.

A batch separation is a first crude separation of proteins from other substances made by forming a slurry with the protein solution and the ion exchange medium and eluting the total volume at once by filtration or centrifugation.

The column method is used for the separation of proteins based on their net charge . Negatively charged proteins can be separated on positively charged solid phase, e.g. DEAE-cellulose or DEAE Sephadex. To separate positively charged proteins a negatively charged solid phase is used, e.g. carboxymethyl cellulose.

The column is packed with the appropriate support medium (stationary phase). The ion exchange medium is the stationary phase and an aqueous solution with increasing salt concentration or decreasing pH is used as the mobile phase. The rate of elution of the proteins off the column is based on their number of charges and the signs (+ or -)of those charges.

Early stationary phases were such compounds as hyroxyapatite and alumina. Most materials used now are ion exchange resins. These resins are strongly basic quaternary amines, e.g. triethylaminoethyl groups (TEAE) or weakly basic groups of diethylaminoethyl (DEAE) which can have positive charges. Cationic exchange resins bear negatively charged groups. These groups can be sulfonate ions or weakly acidic groups of carboxymethyl or phosphate for example.Thus negatively charged proteins are separated using an anion exchange stationary phase and positively charged proteins are separated using cationic ion exchange stationary phases.

When the pH is changed, weakly acidic or basic groups can increase the selective interaction of the support. The charge on the resin is affected as well as that on the protein. Separation on the column may be improved by altering the pH to increase the retention time on the column and by altering the ionic strength of the mobile phase. The ions in the mobile phase compete for exchange with those on the protein. There is a difference in the kind of ion and the effect it will have on this exchange based on the size of the hydrated ion.

An example of a cationic exchange resin with sulfonate ions interacting with a positively charged protein amino groups is shown in the diagram below. The attraction of the positively charge amino groups for the fixed sulfonate ions is decreased as the sodium concentration is increased. The protein is thus eluted by increasing the salt concentration of the liquid entering the column.

Ion Exchange Chromatography
Practice Exercise 2.6	A good reason for isolating proteins using cation or anion exchange batch chromatography is: no response as a first crude separation from other molecules to determine its isoelectric point as the final step in purification to separate basic from acid proteins

B. Adsorption Chromatography

Adsorption of proteins onto such solid supports as alumina(basic polar), kaolin, bentonite, silica gel(acidic polar), talc, or activated charcoal followed by elution of the protein with various buffers, e.g. phosphate pH 7.0, was an early chromatographic method. The adsorption of proteins onto these compounds results from electrostatic or hydrogen bonding interactions. Solutes in the mobile phase and the proteins compete for the binding sites on the support. Losses of proteins and some denaturation were not totally avoided in this method. Batch methods are used to remove impurities from proteins in solution. Solvents are classified as weak or strong depending upon their absorption. Examples are water and methanol are strong solvents for silica gel, whereas for a non-polar adsorbent such as charcoal, water would be a weak solvent and hexane would be a strong solvent. Times of retention of the protein on the column, which may improve separation, may be altered by changing the composition of a mixture of strong and weak solvents.

C. Partition Chromatography

Separation of proteins by this method is based on the relative solubility of the protein in the stationary phase and the mobile phase. The stationary supports used are diatomaceous earth, Chromosorb P or W. These are coated with a liquid such as dimethylsilicone,trifluorpropylmethyl silicone or proplylene glycol. Selection of the appropriate liquid for the stationary phase coat is important. Polar compounds are most strongly retained by a polar stationary phase and non-polar compounds are retained strongly by a non-polar stationary phase.

Two terms are applied, normal phase and reversed phase partition chromatography. Normal phase uses a polar stationary phase and a non-polar solvent as the mobile phase. In reversed phase, the stationary phase is less polar than the mobile phase. Over time the liquid stationary phase is lost from the support column, particularly if high temperatures are used in the separation. This loss is called "bleeding". Bleeding problems are overcome by using a bonded phase, where the liquid is covalently bound to the solid support. When the mobile phase is a flow of gas through the column, the chromatography is known as gas-liquid chromatography or GLC. When the mobile phase is a flow of liquid through the column it is known as liquid chromatography,(LC). When small columns are used with a tightly packed stationary phase and pressure is applied to move the mobile phase, this is known as high pressure liquid chromatography or HPLC.The isolation (separation or purification) of proteins is done using LC or HPLC and not GLC.

D. Steric Exclusion or Gel Filtration Chromatography

The separation of proteins by this method is based upon their size and to a degree upon their shape and hydration. The stationary supports used are materials that have selective pore sizes that allow some molecules to pass through and which retain larger ones. Among these solid supports are Sephadex(a cross-linked dextran), polyacrylamide gel and agarose. Hydrophilic gels are used for the separation of proteins such as enzymes and antibodies. Proteins may be separated very well from each other by virtue of size and from smaller non-protein molecules. Molecules smaller than the pore size are eluted in the void volume of the column and come off immediately, larger molecules caught in the pore will elute according to their size difference from the pore size. For example, monoclonal immunoglobulin G may be separated very well from other serum proteins by using Sepadex G 200 and a long column. The mobile phase depends upon the compounds to be separated, for proteins this is most often a neutral buffer, e.g. phosphate. In diagram A below, the solution of small and large proteins has been layered on the gel column to begin the separation. The absorbance of the eluate is 0 at that time. As buffer flows through the column as shown in diagram B, the large and small proteins are separated, the eluate begins to contain the larger proteins not witheld by the gel. Light at 280 nm is passed through the eluate and the absorbance is recorded as a measure of the concentration of protein being eluted at that time.

Steric Exclusion Chromatography
Practice Exercise 2.7	A good reason for using steric exclusion chromatography is: no response as a crude separation from other molecules to determine the molecular size to determine the solubility to separate basic from acid proteins

E. Affinity Chromatography or Ligand Chromatography

The specificity of ligand chromatography makes it particularly applicable when a substance which binds the protein is available and when other chromatographic methods do not yield pure protein preparations.When available it makes a good final purification step.

The stationary phase in this type of chromatography contains on its surface an immobilized ligand or binding molecule. The supports may be agarose, cross-linked dextrans, or polyacrylamide. The binding agents depend upon the molecule to be bound. For example, an antigen may be used as the ligand to bind an antibody specific to it. A substrate or cosubstrate analog may be used as the ligand to bind an enzyme specific to it. Elution of the bound protein is made by breaking the binding bond with such agents as guanine hydrochloride, urea, or sulfide or by the addition of a substrate or inhibitor.

Affinity Chromatography
Practice Exercise 2.8	A good reason for isolating proteins using affinity or ligand chromatography is: no response as a first crude separation from other molecules to determine its isoelectric point to separate basic from acid proteins as a good step toward final purification

F. Chromatofocusing

This method uses the isoelectric point (pI) to separate proteins. A weak ion-exchange column is used and the protein(s) are eluted with a buffer called a Polybuffer which creates a linear pH gradient. The proteins eluted at the pH of their isoelectric points. The separation and concentration of the proteins is very effective since focusing of the individual pIs occurs during traverse of the column.

F. Measurement and Evaluation of Chromatographic Separation

As the mobile phase passes through the column and the protein of interest is released into the mobile phase it appears in the effluent from the column. The column effluent passes by a detector which is designed to detect the substance of interest. In the case of proteins the detector has frequently been a densitometer set to measure absorbance of the effluent at 280 nm. A record may be made of the absorbance at specific effluent volumes. This allows the selection of a volume of the liquid which contains most of the protein of interest. To do this small quantities of the effluent are collected by a moving carousel which contains tubes for the collection of effluent. The carousel is set to move the tubes at a specific time period. When the volume of fluid coming off the column is set at a specific rate then the volume of effluent in each tube may be calculated in advance and the tube may be related to the record of absorbance at 280nm. In cases where the detector is not connected to the system, the absorbance at 280 nm is measured for each tube to detect the protein elution. When there are multiple proteins present and several absorbance peaks are found in the effluents, it will be necessary to determine which represents the protein of interest. This may be done by:

1. Take the tube which contains the greatest concentration of protein within each peak(greatest absorbance at 280 nm) and assay for the activity or other unique characteristic of the protein of interest.
2. Subject a molecular weight marker (substance of known molecular weight which has a visible color to it) to steric exclusion column on which the protein was run. Select the protein which has the molecular weight that you believe corresponds to the compound being sought.

The separation of the proteins or resolution is defined as two times the distance between two peaks eluting from the column divided by the sum of the peak widths at halfheight. The peaks are the maxima of the detection of the proteins measured over a period of elution time. This is shown in the diagram below:



Resolution(R)= 2 x (PeakA- PeakB)/ (w@halfheight Peak A + w@halfheight Peak B)   Equation 2.3

The nominator of this equation represents the selectivity of the chromatography. This depends upon the response of the particular protein molecule to the stationary phase. The denominator is the efficiency and is related to the physical parameters of the column and system. The narrower the peak the greater the efficiency.

Data from the methods of extraction and isolation (purification) are kept for analysis and future reference. The data to be kept for each method and type of method used are:

1. Volume of solution
2. Protein Activity or Unit of Unique Characteristic
3. Total Protein Content
4. Specific Activity
5. Yield(%) (taking yield of extract as 100%)
6. Purification (taking specific activity of extract as 100%)

Sample Purification Table

Step)Method	Volume(ml)	Total Activity	Total Protein(mg/ml)	Specific Activity	Yield(%)	Purification
1.0)Extract of Supernatant	500	55	520	0.106	100%	1
2.0)Anionic Exchange Batch	30	36.5	34	1.07	69%	10
3.0)Anionic Exchange Column	6	25.5	16.7	1.53	49%	15
4.0)Steric Exclusion Column	8	21.2	11.3	1.88	40%	18

From: Burden, David W. and Whitney, Donald B. (Biotechnology Proteins to PCR, A course in Streategies and Lab Techniques,Birkhauser Publ.Co. Boston 1995, p89




Evaluation of Chromatographic Purification
Practice Exercise 2.9	Using the data in the purification table below caluclate and select the appropriate specific activity, %yield, and purification for the two column methods. Purification Results Step)Method Volume(ml) Total Activity Total Protein(mg/ml) Specific Activity Yield(%) Purification 1.0)Extract of Supernatant 500 60 520 0.115 100% 1 2.0)Anionic Exchange Column 30 40 35 ? ?% ? 3.0)Steric Exclusion Column 8 25 11.3 ? ?% ? Choose from A.,B.,C.,D.,and/or E. below: Eluate Specific Activity %Yield Purification A. Anion Exchange 0.875 58 7.6 B. Anion Exchange 1.143 67 9.9 C. Steric Exclusion 0.875 42 12 D. Steric Exclusion 2.12 42 19 E. This shows that steric exclusion is a better purification method than anion exchange. no response A and C only B and D only A and D only A, C and E only




Lesson 2:Isolation - The Second Step 2.0 - 5

---

Proteins - Analysis of Structure and Function

4. Electrophoresis

Learning Objectives:


Upon completing this topic and the practice exercises, the student should be able to:
1.Identify the factors that influence electrophoretic mobility.
2.Calculate the electrophoretic mobility
3.Identify a practical use for electrophoresis in protein purification.
4. Justify the use of one type of electrophoresis over another in the determination of isoelectric point, determination of purity, and separation of similar proteins.




Proteins bear both positive and negative charges. When the medium is more acid than the isoelectric point of the protein, the protein bears more positive charge(s) and in alkaline solutions it will bear more negative charge(s). When placed in an electric field the protein will migrate toward the anode in the alkaline solution and toward the cathode in an acid solution. This movement in an electric field is electrophoresis. It is a useful tool in separating and isolating proteins.

The rate of movement depends upon the force exhibited by the electric field, qE, where q is the charge in coulombs on the protein and E is the electric field strength in volts/meter. This force is countered by the frictional force, fv, of movement of the protein through the medium where f is the frictional coefficient related to the size and shape of the protein molecule and the viscosity of the medium. The velocity of movement is v. The movement of the protein represents a balance between these opposing forces.

fv = qE    Equation 2.4

Electrophoresis
Practice Exercise 2.10	The movement of protein molecules in an electric field depends on: no response the molecular size the pH of the buffer the molecular shape the molecular charge all of the above



The electrophoretic mobility, u, can be expressed by the formula below when the charge on the molecule, q, is represented as the product of Z, the number of unit charges and e, the electron or proton charges on the protein.

u = v/E = q/f = Ze/f  Equation 2.5

EXAMPLE: Calculation of electrophoretic mobility

A protein has traveled 3 cm from the point of application in an acrylamide gel 10 cm wide after the current has been on for 75 minutes at a voltage of 200 V in a buffer of pH 8.6

u = (3cm)(10cm)/(75min)(60sec/min)(200V) = 3.33 x 10(-5)cm squared per (Volt)(sec)




Electrophoretic Mobility
Practice Exercise 2.11	What is the electrophoretic mobility of a protein calculated from the following data? A protein has traveled 4 cm from the point of application in an acrylamide gel 5 cm wide and 10 cm long from the anode to the cathode. The current had been on for 80 minutes at 250 Volts with a buffer of pH 8.6. no response 1.66 x 10-5cm squared per (volt)(sec) 3.33 x 10-5cm squared per (volt)(sec) 4.33 x 10-5cm squared per (volt)(sec) 6.66 x 10-5cm squared per (volt)(sec)

Lesson 2:Isolation - The Second Step 2.0 - 6

---

Proteins - Analysis of Structure and Function

Some Terms Given To Electrophoresis

Originally electrophoresis was done using what is called moving boundary electrophoresis. The protein solution was carefully layered on top of a buffer solution and the electrophoresis resulted in moving boundaries of proteins in a liquid medium. These boundaries were detected by a Schlieren optical system. The separation was not always as clear as that obtained by zone electrophoresis. The method is technically difficult and still may be used to determine the electrophoretic mobility of proteins.Zone electrophoresis is the term given to electrophoresis performed on a solid support medium. These kinds of solid supports are discussed below under Electrophoretic Media.

Varied Types of Electrophoresis

Isotachophoresis

Isotachophoresis is a method of electrophoresis in which proteins separate into zones that all migrate at the same rate. The protein zones abut one another but are completely separated from each other(no overlap occurs). The current is carried completely by the ionized proteins. No electroendoosomosis occurs. In this method, the protein sample is placed in a capillary over a solution of ions which are known to travel faster in the current than the proteins or peptides in the sample. The sample in this case is not mixed with buffer or electrolyte solution and the movement of current and the protein relies on the charge on the protein. A solution of ions known to travel slower than any of the proteins in the sample is layered on top. A protein which moves ahead of the protein behind it is not allowed to continue to separate further because this would develop a region with no charge and current would cease.

Isoelectric Focusing

In this type of electrophoresis a pH gradient is used in place of the single pH provided by the buffer in routine or regular electrophoresis. Proteins which are capable of bearing positive or negative charges will migrate in an electric field to the point at which the pH equals its isoelectric point (no net charge). Isoelectric focusing also solves any problem of diffusion following electrophoresis. If a protein should diffuse from its isoelectric point (pH) in any direction it develops a charge and is moved back to the pH of its isoelectric point. This is a very powerful method used to resolve protein mixtures and is useful in determining the isoelectric point of the protein. Proteins with isoelectric points of no greater difference than 0.02 have been separated by this method.

To perform isoelectric focusing polyacrylamide gel is prepared with the addition of ampholytes, an array of polyaminocarboxylic acids, whose isoelectric points vary over a pH range. A strip of filter paper containing a solution of phosphoric acid is placed at the anode and one containing a solution of sodium hydroxide is placed at the cathode. The current is applied and the ampholytes distribute themselves in the gel according to their isoelectric points. The current is halted and the protein sample is applied and the current is restarted. The protein carries an ionic charge the load and the type (positive or negative) depends upon the pH surrounding it. Thus the protein moves with its charge toward the appropriate electrode. The pH at which the protein is found at the conclusion of isoelectric focusing can be determined by using a micro pH electrode at that location. This is the isoelectric point of the protein.



A high voltage power supply is required for isoelectric focusing since voltages of around 2000 are used. In addition the power supply must be able to balance voltage and current to provide a constant power. There is a preparative isoelectric focusing apparatus which allows for separation of larger amounts of a purified protein than does the flat gel analytical method.

Denaturing Gel Electrophoresis

Protein molecules may be separated on the basis of molecular mass by using sodium dodecuyl sulfate (SDS) and beta mercaptoethanol (ME) or dithyothreitol (DTT). This treatment may occur prior to placing the protein on the gel or these agents may be introduced into the gel during its preparation. The SDS is an anionic detergent which disrupts covalent interactions in the protein molecule. It also binds to the polypetides and confers a large negative charge on the molecule which overcomes any native charges. The ME or DDT serve to reduce any disulfide bonds that are present. In this type of electrophoresis, the mobility of most denatured proteins is proportional to the log of their mass. Caution is advised, since this method denatures proteins causing them to lose activity or function.

Two Dimensional Gel Electrophoresis

In this procedure the electrophoresis is performed twice. O'Farrell who first described the method utilized isoelectric focusing in polyacrylamide gel containing beta mercaptoethanol for the first electrophoresis (1st dimension) and denaturing gel with SDS for the second electrophoresis (2nd dimension). The first dimension may be done in gels within a tube or as a slab. After the first electrophoresis is done, a lengthwise portion is cut and placed on the gel to be used for the second electrophoresis. Variations in this type of electrophoresis have been described using SDS in both dimensions. Mixtures of many proteins can be resolved by this method. O'Farrell stated that 1100 spots representing different proteins could be resolved using autoradiography, but less could be seen when using a protein stain or dye. Caution is advised, since this method, when SDS is used in the second dimension, the proteins are denatured which causes them to lose activity or function.

Capillary Electrophoresis

This relatively new method has some features of isotachophoresis, zone electrophoresis, and isoelectric focusing.The protein sample is introduced at the anodic end of a small bore capillary (usually less than 75 micrometers). These columns are about 100 cm long and have a detector at one end. Separation occurs as the protein moves according to its relative partitioning between a moving aqueous phase and a slower-moving micellar phase. Separation may occur in less than a minute. This method allows the detection of 10(-20) mol. protein.

Electrophoresis Terms
Practice Exercise 2.12	What is the advantage of isotachophoresis over zone and moving boundary electrophoresis? A. the electrophoretic medium is easier to work with B. protein migration is unaffected by buffer C. electroendosmosis does not occur D. protein zones have no overlap no response C and D only A and D only A and C only B and C only





Electrophoretic Media

Several types of media have been used to perform electrophoresis. The preferable type is polyacrylamide gel(PAG). Other media such as paper, cellulose acetate, agar and agarose gel have a characteristic of bearing negative charges due to adsorption of hydroxyl ions. These hydroxyl ions remain fixed and the positive ions move freely to the cathode. A net movement of solvent to the cathode results which tends to negate the movement toward the anode of proteins which have isoelectrict points not far removed from the pH of the solvent. This force of solvent movement is known as electroendosmosis. An additional advantage of polyacrylamide gel is that the pore size can be controlled by changing the concentrations of acrylamide and the cross-linker, methylenebisacrylamide. Polyacrylamide gel electrophoresis (PAGE) is often performed using layers of gel that differ in pore size. The layers go from the large pore size at the top of the gel to a smaller pore size gel underneath (separator gel). These layers enable the separation of larger proteins from smaller ones which adds a greater dimension of sizing to the charge characteristics so important to electrophoretic separation. Polyacrylamide gel is somewhat more expensive than agarose gel or cellulose acetate.


 

ELECTROPHORETIC MEDIA

Type	Advantage/Disadvantage
Paper	Disadv.=Long Time Requirement, Electroendoosmosis
Agarose	Advant.High clarity, Disadv=Electroendoosmosis and Pore Size Variability
Acrylamide gels	Adv.=Controlled pore size and unreactive




Electrophoretic Media
Practice Exercise 2.13	What is the main advantage of acrylamide gel electrophoresis? it is inexpensive it allows for sizing of protein molecules electroendosmosis does not occur protein zones have no overlap no response 1 and 3 only 2 and 3 only 3 and 4 only 2,3 and 4 only

Identification of Proteins on Electrophoretic Media

Proteins with few exceptions do not possess enough color to be detected on gels or other support media. It is necessary to use dyes or stains that have an affinity for proteins. A chemical reacton that the protein of interest exhibits may also be used. If one wishes to detect a protein from bone,e.g. alkaline phosphatase, following electrophoretic separation, the gel may be over-laid with the substrate for that enzyme and the reaction produces a color which shows where that enzyme is located on the gel.

General protein dyes are:

• Poinceau S
• Amido Black
• Coomassie Brillian Blue R 250
• Silver Nitrate

The Coomassie Brilliant Blue R 250 and the Silver stain are very useful for polyacrylamide gels since the other dyes stain the polyacrylamide gel, e.g. Ponceau S The sensitivities of detection with two of these dyes are: Coomassie Blue detects as little as 0.1 micrograms protein (about 2 picomoles) and the silver stain detects even less, 0.02 micrograms protein. In the clinical chemistry laboratory Ponceau S is used to stain blood serum proteins separated on agarose or cellulose acetate media.

Other methods used to detect proteins separated by electrophoresis include fluorescence, autoradiography,and elution and mass spectrometry.

Protein Stains on Electrophoretic Media
Practice Exercise 2.14	Which of the following statements are true? Coomassie Blue is the most sensitive stain Ponceau S can not be used on polyacrylamide gels Silver stain is the most sensitive stain Substrate reaction can be used to detect enzymes no response 1 and 2 only 1 and 3 only 2 and 3 only 2,3, and 4 only





Electrophoretic Buffers

The characteristics of the buffer used in preparing the gels and performing the electrophoresis is important for several reasons. Some of these are:
• Buffer pH determines charge on protein
• Buffer pH determines the amount of charge on the protein
• Ionic strength of buffer is inverse to rate of movement of protein
• Ionic strength of buffer is directly related to the sharper resolution of the protein
• High ionic strength of buffer causes heat formation and possible denaturation of proteins

The most commonly used buffers are barbital buffers and tris-boric acid-EDTA buffers.The ionic strengths used range from 0.025 to 0.075 for the barbital buffers to 0.03 to 0.10 for tris-boric acid-EDTA. If buffers of high ionic strength are used for special purposes, the procedure is performed in the cold room or with cold packs on the instrument to reduce the heat.

Electrophoretic Buffers
Practice Exercise 2.15	Which of the following statements are true? Buffer pH determines the charge on the protein High ionic strength usually results in lower rate of protein movement High ionic strength usually results in higher resolution of proteins High ionic strength of buffer causes increase in heat formation no response 1 and 2 above 2 and 3 above 2,3 and 4 above all of the above

Characteristics of Electrophoretic Methods
Practice Exercise 2.16	Match the principal characteristic with the method. Isoelectric Focusing A. denatures protein Capillary Electrophoresis B. determine pI Two Dimensional Gel Electrophoresis C. fast & good resolution SDS Gel Electrophoresis D. resolves large numbers of proteins no response 1-B,2-C,3-D,4-A 1-A, 2-B, 3-C, 4-D 1-C, 2-D, 3-B, 4-C 1-D, 2-A, 3-D, 4-B





Lesson 2:Isolation - The Second Step 2.0 - 6

---

---

Final Instructions

Press Button below for your score. After completing Lesson 2.0, including all practice exercises, press the "Submit..." button for Lesson 2.0 credit. After you press "Submit..."it is possible Netscape may tell you it is unable to connect because of unusually high system demands. If you receive no error message on your submission you're OK. But, if Netscape gives you an error message after you press the "Submit..." button, wait a moment and resubmit or consult the attendant. End of Lesson 2.0-Isolation - The Second Step In Proteins-Analysis of Structure and Function Go to Lesson3




Lesson 2:Isolation - The Second Step 2.0 - 7

---