1. Protein Purification Fig. 5-CO, p.113
Column chromatography is widely used in working with proteins.
Fig. 5-CO, p.113

2. Protin Purificartion & Characterizing Techniques
1. How Do We Extract Pure Proteins from Cells?
Disruption of cells is the first step in protein purification. The various parts of cells can be separated by centrifugation. This is a useful step because proteins tend to occur in given organelles. High salt concentrations will precipitate groups of proteins, which are then further separated by chromatography and electrophoresis.

3. 2. What Is Column Chromatography?
Two of the most important methods for separating amino acids, peptides, and proteins are chromatography and electrophoresis. The various forms of chromatography rely on differences in charge, polarity, or size of the molecules to be separated, depending on the application.

4. 3. What Is Electrophoresis?
In electrophoresis, differences in charge and in size are the criteria for separation. The sieving action of gel slabs is used in conjunction with the charge on proteins to achieve separation. The electrophoretic mobilities of proteins can be used to estimate their molecular weights.

5. 4. How Do We Determine the Primary Structure of a Protein?
Determination of the N-terminal and C-terminal amino acids of proteins depends on the use of these separation methods after the ends of the molecule have been chemically labeled. Selective cleavage of the protein into peptides by enzymatic or chemical hydrolysis produces fragments of manageable size for sequencing.

6. PROTEINS & PEPTIDES MUST BE PURIFIED PRIOR TO ANALYSIS
Highly purified protein is essential for determination of its amino acid sequence. Cells contain thousands of different proteins, each in widely varying amounts. The isolation of a specific protein in quantities sufficient for analysis presents a challenge that may require multiple successive purification techniques.

7. Table 5-1, p.114

8. Dialysis.
Protein molecules (red) are retained within the dialysis bag, whereas small molecules (blue) diffuse

9. FIGURE 5.1 Differential centrifugation is used to separate cell components. As a cell homogenate is subjected to increasing g forces, different cell components end up in the pellet.
Fig. 5-1, p.115

10. Gel-Filtration Chromatography
FIGURE 5.2 An example of column chromatography. A sample containing several components is applied to the column. The various components travel at different rates and can be collected individually.
Fig. 5-2, p.116

11. 33
FIGURE 5.3 The repeating disaccharide unit of agarose, which is used for column chromatography.
Fig. 5-3, p.117

12. FIGURE 5. 5 Gel-filtration chromatography
FIGURE 5.5 Gel-filtration chromatography. (a) Larger molecules are excluded from the gel and move more quickly through the column. Small molecules have access to the interior of the gel beads, so they take a longer time to elute.
Fig. 5-5a, p.118

13. FIGURE 5. 5 Gel-filtration chromatography
FIGURE 5.5 Gel-filtration chromatography. (b) V0 is the void volume, the volume of elution for a molecule excluded from the gel bead. Ve is the elution volume for a particular molecule that can enter the bead. Vt is the total volume, the elution volume for a very small molecule that enters the bead unhindered.
Fig. 5-5b, p.118

14. Affinity Chromatography
The principle of affinity chromatography. In a mixture of proteins, only one (designated P1) will bind to a substance (S) called the substrate. The substrate is attached to the column matrix. Once the other proteins (P2 and P3) have been washed out, P1 can be eluted, either by adding a solution of high salt concentration or by adding free S.
Fig. 5-6, p.118

15. Affinity Chromatography Affinity chromatography of concanavalin A (shown in yellow) on a solid support containing covalently attached glucose residues (G).
The plant protein concanavalin A can be purified by passing a crude extract through a column of beads containing covalently attached glucose residues.
Concanavalin A binds to such a column because it has affinity for glucose, whereas most other proteins do not. The bound concanavalin A can then be released from the column by adding a concentrated solution of glucose.

16. Ion-Exchange Chromatography
Ion-Exchange Chromatography. This technique separates proteins mainly according to their net charge.

17. Ion-Exchange Chromatography.
FIGURE 5.8 Operation of a cation-exchange column, separating a mixture of aspartate, serine, and lysine. (a) The cation-exchange resin in the beginning, Na+ form. (b) A mixture of aspartate, serine, and lysine is added to the column containing the resin. (c) A gradient of the eluting salt (for example, NaCl) is added to the column. Aspartate, the least positively charged amino acid, is eluted first. (d) As the salt concentration increases, serine is eluted. (e) As the salt concentration is increased further, lysine, the most positively charged of the three amino acids, is eluted last.
Fig. 5-8, p.120

18. FIGURE 5.7 (a) Cation-exchange resins
Fig. 5-7a, p.119

19. FIGURE 5.7 (b) anion-exchange resins commonly used for biochemical separations.
Fig. 5-7b, p.119

20. FIGURE 5. 9 Ion-exchange chromatography using a cation exchanger
FIGURE 5.9 Ion-exchange chromatography using a cation exchanger. (a) At the beginning of the separation, various proteins are applied to the column. The column resin is bound to Na+ counterions (small red spheres). (b) Proteins that have no net charge or a net negative charge pass through the column. Proteins that have a net positive charge stick to the column, displacing the Na+. (c) An excess of Na+ ion is then added to the column. (d) The Na+ ions outcompete the bound proteins for the binding sites on the resin, and the proteins elute.
Fig. 5-9, p.120

21. Chromatographic Separations
Partition molecules between two phases, one mobile and the other stationary. For separation of amino acids or sugars, the stationary phase, or matrix, may be a sheet of filter paper (paper chromatography) or a thin layer of cellulose, silica, or alumina (thin-layer chromatography);

22. Electrophoresis
Electrophoresis: the process of separating compounds on the basis of their electric charge & size
electrophoresis of amino acids can be carried out using paper, starch, agar, certain plastics, and cellulose acetate as solid supports
in paper electrophoresis, a paper strip saturated with an aqueous buffer of predetermined pH serves as a bridge between two electrode vessels.

23. a sample of amino acids is applied as a spot (the origin) on the solid support strip
an electric potential is applied to the electrode vessels and amino acids migrate toward the electrode with charge opposite their own
molecules with a high charge density move faster than those with a low charge density
molecules at their isoelectric point remain at the origin
after separation is complete, the strip is dried and developed to make the separated amino acids visible

24. FIGURE 5. 10 The experimental setup for gel electrophoresis
FIGURE 5.10 The experimental setup for gel electrophoresis. The samples are placed on the left side of the gel. When the current is applied, the negatively charged molecules migrate toward the positive electrode.
Fig. 5-10, p.121

25. Polyacrylamide Gel Electrophoresis.

26. FIGURE 5. 11 Separation of proteins by gel electrophoresis
FIGURE 5.11 Separation of proteins by gel electrophoresis. Each band seen in the gel represents a different protein. In the SDS–PAGE technique, the sample is treated with detergent before being applied to the gel. In isoelectric focusing, a pH gradient runs the length of the gel.
Fig. 5-11, p.121

27. Electrophoretic Analysis of a Protein Purification.

28. FIGURE 5.12 A plot of the log of the molecular weight versus the relative electrophoretic mobility.
Fig. 5-12, p.121

29. Two-Dimensional Gel Electrophoresis.
FIGURE 5.13 Two-dimensional electrophoresis. A mixture of proteins is separated by isoelectric focusing in one direction. The focused proteins are then run using SDS–PAGE perpendicular to the direction of the isoelectric focusing. Thus the bands that appear on the gel have been separated first by charge and then by size.
Fig. 5-13, p.122

30. molecular weights of sample proteins
S value (Svedberg units)
Molecular weight
Pancreatic trypsin inhibitor 1
6,520
Cytochrome c
1.83
12,310
Ribonuclease A
1.78
13,690
Myoglobin
1.97
17,800
Trypsin
2.5
23,200
Carbonic anhydrase
3.23
28,800
Concanavlin A
3.8
51,260
Malate dehydrogenase
5.76
74,900
Lactate dehydrogenase
7.54
146,200

31. How Do We Determine the Primary Structure of a Protein?

32. FIGURE 5.14 The strategy for determining the primary structure of a given protein. The amino acid sequence can be determined by four different analyses performed on four separate samples of the same protein.
Fig. 5-14, p.123

33. (a) Trypsin is a proteolytic enzyme, or protease, that specifically cleaves only those peptide bonds in which arginine or lysine contributes the carbonyl function. (b) The products of the reaction are a mixture of peptide fragments with C-terminal Arg or Lys residues and a single peptide derived from the polypeptide’s C-terminal end.
Fig. 5-16, p.124

34. Cleavage of proteins by enzymes
Cleavage of proteins by enzymes. Chymotrypsin hydrolyzes proteins at aromatic amino acids.
Fig. 5-17, p.125

35. Chemical Cleavage Cyanogen bromide
FIGURE 5.18 Cleavage of proteins at internal methionine residues by cyanogen bromide.
Fig. 5-18b, p.125

36. Edman reagent is phenylisothiocyanate

37. Specific cleavage of polypeptides
Cleavage site
Reagent
Chemical cleavage
Carboxyl side of methionine residues
Cyanogen bromide
Enzymatic cleavage
Carboxyl side of lysine and arginine residues
Trypsin
Carboxyl side of arginine
Thrombin
Carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine
Chymotrypsin
Amino side of C-terminal amino acid (not arginine, lysine, or proline)
Carboxypeptidase A

38. FIGURE 5.15 HPLC chromatogram of amino acid separation.
Fig. 5-15, p.123

39. FIGURE 5.18 Cleavage of proteins at internal methionine residues by cyanogen bromide.
Fig. 5-18a, p.125

40. FIGURE 5.19 Use of overlapping sequences to determine protein sequence. Partial digestion was effected using chymotrypsin and cyanogen bromide. For clarity, only the original N-terminus and C-terminus of the complete peptide are shown.
Fig. 5-19, p.126

41. END