Ionic Charge

All proteins have charges on them as a result of amino acid side chains such as aspartate, glutamate, histidine, lysine and arginine. The net charge on a given protein depends on its exact composition, and on the pH. Consequently at a given pH different proteins will have different net charges, and a shift in pH will change this value for each protein, though for all them it will become more negative for a higher pH, and more positive for a lower pH.

Although it is possible to exploit the charge differences by electrophoretic techniques, this has rarely been fully successful in preparative (as opposed to analytical) separations of proteins. The main problems have been in the design of reliable and safe equipment; there are some useful systems, but usually they will only be used when all else has failed. Ionic charge separations are carried out by ion exchange chromatography, which has been the most successful and widely used method of protein separation since its introduction some 40 years ago. Anion exchange columns have positive charges that attract negatively charged proteins, and at neutral pH most native proteins are negatively charged. For the minority of positively charged (high isoelectric point) proteins, cation exchangers are used. Samples are applied in a buffer that has low salt content (low ionic

Table 1 Results of a typical ammonium sulfate fractionation procedure in purifying an enzyme. Percentages are given as a percentage of saturation with ammonium sulfate. Specific activity is in units of enzyme activity per milligram of protein. Although the degree of purification is not high (2.1-fold), it is useful to note that much of the nonprotein material stays in the supernatant, and the precipitated fraction containing the enzyme can be dissolved in a much smaller volume than the starting extract. Results are also given for a second trial, in which recovery of activity has been sacrificed for a higher degree of purification

Table 1 Results of a typical ammonium sulfate fractionation procedure in purifying an enzyme. Percentages are given as a percentage of saturation with ammonium sulfate. Specific activity is in units of enzyme activity per milligram of protein. Although the degree of purification is not high (2.1-fold), it is useful to note that much of the nonprotein material stays in the supernatant, and the precipitated fraction containing the enzyme can be dissolved in a much smaller volume than the starting extract. Results are also given for a second trial, in which recovery of activity has been sacrificed for a higher degree of purification

Fraction

Volume

Amount of

Amount of

Specific

Purification

Recovery

(mL)

protein (mg)

enzyme (units)

activity

(x-fold)

(%)

Extract

500

6000

380

0.063

1

100

0-50%

20

750

20

50-60%

45

2400

310

0.130

2.1

82

60-70%

35

1600

70

70% supernatant

540

1000

0

0-55%

40

1500

90

55-60%

35

1300

220

0.169

2.7

53

60% supernatant

535

3100

70

Figure 1 Example of protein separation using anion exchange chromatography. (A) A diagram of the basic principles of the equipment. The amount of protein emerging from the column is monitored by detecting the absorbance of light in the ultraviolet region (280 nm or 215 nm) due to proteins, and separate fractions are collected automatically. (B) The elution pattern. Some proteins did not bind to this adsorbent, being positively charged at this pH. The elution position of a specific protein is indicated by the shaded peak.

Figure 1 Example of protein separation using anion exchange chromatography. (A) A diagram of the basic principles of the equipment. The amount of protein emerging from the column is monitored by detecting the absorbance of light in the ultraviolet region (280 nm or 215 nm) due to proteins, and separate fractions are collected automatically. (B) The elution pattern. Some proteins did not bind to this adsorbent, being positively charged at this pH. The elution position of a specific protein is indicated by the shaded peak.

strength), and proteins of the opposite charge to the column are adsorbed. Proteins are generally eluted by increasing the ionic strength with gradual salt addition to the buffer, so that proteins of successive adsorbing strengths elute as the salt concentration goes up. This enables relatively high resolution of separate protein components, giving a high degree of purification. There are many commercial adsorbents and equipment to use them with. The type to be used will depend very much on the purpose; 'high performance' systems are expensive to operate and may not be suitable for commercial production of the protein, but most suited to research and development. Speed may be desirable, or may be of little concern; there are materials for all needs. An example of the separation of a fairly complex mixture of proteins on a 'moderate performance' adsorbent is shown in Figure 1.

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