Lecture 5: 8/31. CHAPTER 5 Techniques in Protein Biochemistry

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1 Lecture 5: 8/31 CHAPTER 5 Techniques in Protein Biochemistry


3 Chapter 5 Outline

4 The proteome is the entire set of proteins expressed and modified by a cell under a particular set of biochemical conditions. Unlike the genome, the proteome is not an unvarying characteristic of the cell.

5 Protein purification requires a test or an assay. An assay determines whether the protein of interest is present in the cell.

6 An assay for the enzyme lactate dehydrogenase is based on the fact that a product of the reaction, NADH, can be detected spectrophotometrically.

7 Protein purifications are monitored in part by determining the specific activity of the protein being purified. In the case of an enzyme purification, specific activity is the ratio of enzyme activity to protein concentration. Specific activity should increase with each step of the purification procedure.


9 Techniques for Protein Purification Differential centrifugation Cells are disrupted to form a homogenate, which is a mixture of all of the components of the cell, but no intact cells. The homogenate is then centrifuged at low speed to yield a pellet consisting of nuclei and a supernatant. This supernatant is then centrifuged at a higher centrifugal force to yield another pellet and supernatant. This process, called differential centrifugation, is repeated several more times to yield a series of pellets enriched in various cellular materials and a final supernatant called the cytosol.

10 Differential centrifugation

11 Salting in and Salting out Salting out takes advantage of the fact that the solubility of proteins varies with the salt concentration. Most proteins require some salt to dissolve in water, a process called salting in. As the salt concentration is increased, different proteins will precipitate at different salt concentrations, a process called salting out. Principal of Salting Out: Competition between the salt ions and protein for water results in protein precipitation.

12 The dependency of protein solubility on salt concentration

13 The salt can be removed from a protein solution by dialysis. The protein solution is placed in a cellophane bag with pores too small to allow the protein to diffuse, but big enough to allow the salt to equilibrate with the solution surround the dialysis bag.

14 Dialysis Protein molecules (red) are retained within the dialysis bag, whereas small molecules (blue) diffuse into the surrounding medium.

15 Molecular exclusion chromatography (gel filtration chromatography) Principal: Molecular exclusion chromatography (gel filtration chromatography) allows the separation of proteins on the basis of size. A glass column is filled with porous beads. When a protein solution is passed over the beads, large proteins cannot enter the beads and exit the column first. Small proteins can enter the beads and thus have a longer path and exit the column last.

16 Gel filtration chromatography

17 Which of the following techniques is most useful for fractionating a heterogeneous protein mixture by size A. Affinity chromatography B. Edman degradation C. Gel-filtration chromatography D. Ion-exchange chromatography E. Isoelectric focusing 0% 0% 0% 0% 0%

18 Ion exchange chromatography Principal: Ion exchange chromatography allows separation of proteins on the basis of charge. The beads in the column are made so as to have a charge. When a mixture of proteins are passed through the column, proteins with the same charge as on the column will exit the column quickly. Proteins with the opposite charge will bind to the beads, and are subsequently released by increasing the salt concentration or adjusting the ph of the buffer that is passed through the column.

19 Ion exchange chromatography This technique separates proteins mainly according to their net charge

20 Affinity chromatography Principal: Some proteins have a high affinity for specific chemicals or chemical groups. This characteristic of protein can be utilized for protein purification. Beads are made with the specific chemical attached. A protein mixture is passed through the column. Only protein with affinity for the attached group will be retained. The bound protein is then released by passing a solution enriched in the chemical to which the protein is bound.

21 Affinity chromatography

22 High pressure liquid chromatography (HPLC) The resolving power of any chromatographic technique is related to the number of potential sites of interaction between the protein and the column beads. Very fine beads allow more interactions and thus greater resolving power, but flow rates through such columns are too slow. High pressure liquid chromatography (HPLC) uses very fine beads in metal columns and high pressure pumps to move the liquid through the column. Because of the increased number of interaction sites, the resolving power of HPLC is greater than normal columns.

23 High pressure liquid chromatography (HPLC)

24 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE Principal: Proteins will migrate in an electrical field because they are charged. When the migration occurs in a gel, the process is called gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) allows accurate determination of mass. SDS denatures proteins and, for most proteins, 1 molecule of SDS binds for every two amino acids. Thus, proteins have the same charge to mass ratio and migrate in the gel on the basis of mass only.

25 Which of the following is necessarily characterized by a covalent linkage A. Base-pairing interaction B. Disulfide bond C. Hydrogen bond D. Van der Waals interaction 0% 0% 0% 0%

26 The relative mobility of different proteins during SDS-polyacrylamide gel electrophoresis is primarily determined by which property of the proteins A. Antigenicity B. Hydrophobicity C. Isoelectric point D. Mass 0% 0% 0% 0%

27 Polyacrylamide gel electrophoresis

28 The staining of proteins after electrophoresis Proteins separated by SDS PAGE are visualized by staining the gel with dyes such as Coomassie blue.

29 Isoelectric focusing Principal: Isoelectric focusing allows separation of proteins in a gel on the basis of their relative amounts of acidic and basic amino acids. If a mixture of proteins is placed in a gel with a ph gradient and an electrical field is applied, proteins will migrate until they reach their isoelectric point (pi), the ph at which they have no net charge.

30 The principle of isoelectric focusing A ph gradient is established in a gel before the sample has been loaded. (A) The sample is loaded and voltage is applied. The proteins will migrate to their isoelectric ph, the location at which they have no net charge. (B) The proteins form bands that can be excised and used for further experimentation.

31 Two dimensional gel electrophoresis (2D gel) In two dimensional gel electrophoresis, proteins are separated in one direction by isoelectric focusing. This gel is then attached to an SDS PAGE gel and electrophoresis is performed at a 90 angle to the direction of the isoelectric focusing separation.

32 Two dimensional gel electrophoresis (A) A protein sample is initially fractionated in one direction by isoelectric focusing as described in Figure 5.10.The isoelectric focusing gel is then attached to an SDSpolyacrylamide gel, and electrophoresis is performed in the second direction, perpendicular to the original separation. Proteins with the same pi value are now separated on the basis of mass.

33 Two dimensional gel electrophoresis

34 Alterations in protein levels detected by two dimensional gel electrophoresis.

35 The effectiveness of a purification scheme is measured by calculating the specific activity after each separation technique. SDS PAGE allows a visual evaluation of the purification scheme.


37 Effectiveness of the purification techniques Electrophoretic analysis of a protein purification

38 What physical differences among proteins allow their purifications A. Solubility B. Size C. Charge D. Binding affinity E. All of the above 20% 20% 20% 20% 20%

39 Gradient centrifugation The estrogen receptor binds the steroid hormone estradiol tightly and with great specificity. The estrogen receptor has no enzymatic activity, but can be purified by immunological techniques and the use of gradient centrifugation.

40 Ultracentrifugation can be used to examine proteins. When subjected to a centrifugal force, the rate of movement of the particle is defined by the sedimentation coefficient, s. Where m = mass, = the partial specific volume (the reciprocal of the particle density), ρ =density of the medium, and = the frictional coefficient of the particle. Sedimentation coefficients are usually expressed as Svedberg units (S) equal to s. The smaller the S value (i.e. smaller the molecular weight), the slower the protein moves in a centrifugal field.

41 Sedimentation coefficients are usually expressed as Svedberg units (S) equal to s. The smaller the S value (i.e. smaller the molecular weight), the slower the protein moves in a centrifugal field.


43 A density gradient is formed in a centrifuge tube, and a mixture of proteins in solution is placed on top of the gradient. To identify the estradiol receptor, the protein mixture is first incubated with radioactive estradiol, which is readily detected. Only the estradiol receptor will bind to the steroid. Moreover, the steroid alone is too small to be influenced by the centrifugal force. After the centrifugation is complete, a small hole is made in the bottom of the centrifuge tube and portions of the gradient are collected and tested for radioactivity.

44 Zonal centrifugation, aform a density gradient

45 Gradient centrifugation analysis of the estradiol receptor complex

46 An antibody is a protein synthesized in response to the presence of a foreign substance called an antigen. The antibody recognizes a particular structural feature on the antigen called the antigenic determinant or epitope.

47 Antibody structure

48 Any antibody producing cell synthesizes antibodies that recognize only one epitope. Each antibody producing cell thus synthesizes a monoclonal antibody. Any antigen may have multiple epitopes. The antibodies produced to the antigen by different cells are said to be polyclonal.


50 Immortal cell lines producing monoclonal antibodies can be generated by fusing normal antibody producing cells with cells from a type of cancer called multiple myeloma. A monoclonal cell line is isolated by screening for the antibody of interest.

51 The preparation of monoclonal antibodies

52 A monoclonal antibody for the estrogen receptor can be isolated by searching for cell lines that produce an antibody that binds to the receptor. If an antibody for the receptor is present, it will bind to the receptor and alter the sedimentation constant of the receptor.

53 Alteration of the sedimentation profile when antibody binds to the receptor protein

54 Once the monoclonal cell line is isolated, the antibody can be used to purify the estrogen receptor.

55 Once the monoclonal cell line is isolated, the antibody can be used to purify the estrogen receptor. Purification by immunoprecipitation (IP)


57 Purification by immunoprecipitation

58 Enzyme linked immunosorbent Assay (ELISA) Antibodies are used as a reagent to determine the amount of a protein or other antigen present. Enzyme linked immunosorbent Assay (ELISA) quantifies the amount of protein present because the antibody is linked to an enzyme whose reaction yields a readily identified colored product.

59 Indirect ELISA and sandwich ELISA (a clinically used assay)



62 In western blotting or immunoblotting, proteins are separated in an SDS PAGE gel, transferred to a sheet of polymer, and then stained with a fluorescent antibody.

63 Western blotting Proteins on an SDS polyacrylamide gel are transferred to a polymer sheet and stained with fluorescent antibody. The fluorescent antibody is excited by light and the band corresponding to the protein to which the antibody binds is visualized with an appropriate detector.


65 Determination of amino acid composition. A key step in understanding protein function is to determine the primary structure, or the amino acid sequence, of the protein. A preliminary step is to determine the amino acid composition of the protein. The protein is hydrolyzed, and the constituent amino acids are separated on an ion exchange column. The amino acids are visualized by reaction with fluorescamine.

66 Fluorescent derivatives of amino acids. Fluorescamine reacts with the amino group of an amino acid to form a fluorescent derivative

67 Determination of amino acid composition

68 Edman degradation The amino acid sequence can be determined by Edman degradation. The protein is exposed to phenyl isothiocyanate (PTH), which reacts with the N terminal amino acid to form a PTH derivative. The PTH amino acid can be released without hydrolyzing the remainder of the protein, and the degradation is subsequently repeated.

69 The Edman degradation



72 Because the reactions of the Edman degradation procedure are not 100% effective, it is not possible to sequence polypeptides longer than 50 amino acids. In order to sequence the entire protein, the protein is chemically or enzymatically cleaved to yield peptides of fewer than 50 amino acids. The peptides are then ordered by performing a different cleavage procedure in order to generate overlap peptides.



75 Mass Spectrometry Can Be Used to Determine a Protein s Mass, Identity, and Sequence Two mass spectrometry techniques can be used to determine a proteins mass: matrix assisted laser desorption (MALDI) and electrospray ionization (ESI). In MALDI, proteins are precipitated onto a matrix and a laser flash releases negatively charged ions. Time of flight (TOF) analysis is used to measure how rapidly the ions move toward a detector. Only picomoles or femtomoles of a protein are required for MALDI TOF analysis.

76 MALDI TOF mass spectrometry (1) The protein sample, embedded in an appropriate matrix, is ionized by the application of a laser beam. (2) An electric field accelerates the ions through the flight tube toward the detector. (3) The lightest ions arrive first. (4) The ionizing laser pulse also triggers a clock that measures the time of flight (TOF) for the ions.

77 MALDI TOF mass spectrum of insulin and lactoglobulin

78 Mass spectrometry allows determination of a protein s identity. For instance, an unknown protein visible in a two dimensional gel can be removed from the gel. The protein is then cleaved in some fashion and subjected MALDI TOF, revealing a series of peptides with known masses. These peptide masses are then compared to proteins in a data base that are electronically cleaved by a computer using the same cleavage technique used to generate the protein fragments.

79 Peptide sequencing by tandem mass spectrometry A proteins sequence can be determined with the use of tandem mass spectrometry.

80 1. Primary structures from different proteins can be compared to infer knowledge about structure and function. 2. Primary structure comparison of similar proteins from different species provides information about evolution. 3. Primary structure can be searched for internal repeats that may yield information on the history of the individual protein.

81 Repeating motifs in a protein chain

82 4. Primary structure can reveal the presence of amino acid sequences that regulate protein function and location. 5. Primary structure can provide insight into the molecular basis of disease. 6. Primary structure can be used as a guide to explore nucleic acid information.