Estimation of the Maximum Migration Distance and Recognition of Size Isomers

With increasing times of electrophoresis under non-denaturing conditions, the migration of proteins in a PA gradient gel gradually decreases (Figures 9-11).

Figure 9 Time-dependent migration patterns of marker proteins and carbonic anhydrase (EC 4.2.1.1) (iso)enzymes from mammalian erythrocytes. Lanes 1 and 6, marker proteins. Lanes 2-5; carbonic anhydrases from (2) bovine, (3) human, (4) rabbit and (5) canine. Mol mass of marker proteins: ovalbumin (45000), bovine serum albumin (67000), lactate dehydrogenase (140000), catalase (232 000), ferritin (440000) and thyroglobulin (669000). Linear PA 4-30% T gradient (acrylamide-Bis = 24 : 1), 300 V per 73 mm of gel length, 5°C. Running times: 2, 8 and 16 h. Gel and electrode buffer: 90 mmol L~1 Tris, 80 mmol L~1 boric acid, 1.25 mmol L~1 EDTA-Na2-H2O, pH 8.4. Protein staining with Coomassie brilliant blue. Enzyme preparations from Sigma, Munich, Germany. Reproduced with permission from Rothe (1991).

Figure 9 Time-dependent migration patterns of marker proteins and carbonic anhydrase (EC 4.2.1.1) (iso)enzymes from mammalian erythrocytes. Lanes 1 and 6, marker proteins. Lanes 2-5; carbonic anhydrases from (2) bovine, (3) human, (4) rabbit and (5) canine. Mol mass of marker proteins: ovalbumin (45000), bovine serum albumin (67000), lactate dehydrogenase (140000), catalase (232 000), ferritin (440000) and thyroglobulin (669000). Linear PA 4-30% T gradient (acrylamide-Bis = 24 : 1), 300 V per 73 mm of gel length, 5°C. Running times: 2, 8 and 16 h. Gel and electrode buffer: 90 mmol L~1 Tris, 80 mmol L~1 boric acid, 1.25 mmol L~1 EDTA-Na2-H2O, pH 8.4. Protein staining with Coomassie brilliant blue. Enzyme preparations from Sigma, Munich, Germany. Reproduced with permission from Rothe (1991).

Migration of globular proteins comes to an end when the maximum pore size of a gel region equals their own size. The corresponding migration distance is called the maximum migration distance (Dmax (mm)). The maximum migration distance can be obtained from a number of time-dependent protein migrations (D (mm)) (Figures 9 and 10) which are directly measured on the gel after proteins have been visualized following electrophoretic separation (Table 2). To obtain the maximum migration distance of a certain protein, the following mathematical approximation procedure can be applied: the migration distances are double-logarithmized (ln(ln D)) and plotted versus the reciprocal of the square root of electrophoretic migration time, 1/t1/2 (t (h)). This results in a straight line (Figure 11) whereby the transformed migration values (ln(ln D)) and the transformed times of electrophoresis (t~1/2) are interrelated by the equation:

becomes:

and:

where a and b are the slope and the intercept of the corresponding straight line. The equation predicts that at very high values of t, t~1/2 reaches zero. This means that the maximum migration of a protein (Dmax (mm)) can be taken from the intercept of the straight line with the ordinate in a plot of ln(ln D) versus t~1/2 provided protein migrations were larger than 2 mm and a sufficient number of different migration distances are registered. Letting t approximate to infinity means that eqn [5]

A plot of ln(ln D) versus t~1/2 can also be used to distinguish size isomers from charge isomers. Equally sized but differently charged forms of an enzyme or protein system are recognized by the fact that the straight line of each enzyme form intersects at the same point on the ln(ln D) axis as is for example the case with mammalian carbonic anhydrase (cf. Figure 11) and mammalian lactate dehydrogenase. On the other hand, migration of charge isomers should result in lines of equal slope. Proteins differing in charge and size, however, give straight lines with both different slopes and intercepts.

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