Determination of Migration Velocities

The migration velocity of a protein migrating in an electrophoretic support medium can be obtained by computing the quotient of the difference in the distance migrated between two consecutive time

10 11 12 13 14

In Mr

Figure 12 Calibration lines to calculate the molecular mass (M) and Stokes radius (RS) of five carbonic anhydrase isoenzymes. The logarithm of the maximum migration distance (ln Dmax) correlates linearly to the logarithm of the mol mass (ln Mr) and the logarithm of the Stokes radius (ln RS), respectively. CA, Carbonic anhydrase (average ln Dmax of isozymes I-V); OVA, ovalbumin; BSA; bovine serum albumin; LDH, lactate dehydrogenase; CAT, catalase; FER, ferritin; THY, thyroglobulin. The calculated mol masses and Stokes radii are listed in Tables 3 and 4.

10 11 12 13 14

In Mr

Figure 12 Calibration lines to calculate the molecular mass (M) and Stokes radius (RS) of five carbonic anhydrase isoenzymes. The logarithm of the maximum migration distance (ln Dmax) correlates linearly to the logarithm of the mol mass (ln Mr) and the logarithm of the Stokes radius (ln RS), respectively. CA, Carbonic anhydrase (average ln Dmax of isozymes I-V); OVA, ovalbumin; BSA; bovine serum albumin; LDH, lactate dehydrogenase; CAT, catalase; FER, ferritin; THY, thyroglobulin. The calculated mol masses and Stokes radii are listed in Tables 3 and 4.

Table 3 Calculated molecular mass of marker proteins and mammalian carbonic anhydrase (iso)enzymes and calculation of percentage of deviation of the calculated values from the literature

(Mr (gmor'))a

(f/fo)

Calculated mol mass (Mr)

In Mr Mr" Deviationc

(%)

ln Dmax

Ovalbumin

43000

10.6690

1.18

10.8234

50181

# 16.7

4.6563

Bovine serum albumin

67000

11.1125

1.34

11.2981

80668

#20.4

4.3537

l-lactate dehydrogenase

140000

11.8494

11.7695

129249

-7.7

4.0532

Catalase

232000

12.3545

1.27

12.0356

168653

-27.3

3.8836

Ferritin

440000

12.9945

1.40

12.7717

352110

-20

3.4144

Thyroglobulin

669000

13.4135

13.6949

886379

#32

2.8259

Carbonic anhydrase

Bovine I

10.5233

37171

4.8476

Bovine II

10.4904

35968

4.8686

Bovine/rabbit III

38000

10.5373

37695

4.8387

Rabbit IV

10.5775

39241

4.8131

Canine/human V

29700

10.5462

38032

4.8330

Arithmetic mean

10.5346

37594

4.8404

a Literature values.

bThe molecular mass of bovine carbonic anhydrase as estimated by sequence analysis was reported to be 28980 while that of the enzyme from mouse was found to be 29068.

cThe molecular sizes calculated are compared with the literature data and the percentage deviation indicated. Reproduced with permission from Rothe (1991).

intervals during electrophoresis, and the correspond- Eqn [16] summarizes this procedure:

ing time difference

v (mm s_i) =

(D1

-Do)x

(ti - to)

v (mm s_1) =

(D2

-Di)x

(t2 - ti)

v (mm s_1) =

(D3

-D2)x

(t3 - t2)

where Dn (mm) equals the migration distance of a protein at a time tn (s) and Dm (mm) equals its migration distance at a time tm (s) where tn > tm (Figure 13).

Table 4 Calculated Stokes radius of marker proteins and mammalian carbonic anhydrase (iso)enzymes and calculation of percentage of deviation of calculated values from the literature

Protein

Stokes radius (Rs (nm))a

ln Rs

Calculated Stokes radius (RS)

ln RS RS Percentage (nm) deviation"

ln Dmax

Ovalbumin

3.05

- 19.6081

- 19.5992

3.08

#0.9

4.6563

Bovine serum albumin

3.55

- 19.4563

- 19.4285

3.65

#2.8

4.3537

l-Lactate dehydrogenase

4.20

- 19.2881

- 19.2590

4.32

#2.9

4.0532

Catalase

5.25

- 19.0650

- 19.1634

4.76

-9.3

3.8836

Ferritin

6.10

- 18.9150

- 18.8987

6.20

#1.6

3.4144

Thyroglobulin

8.50

- 18.5832

- 18.5668

8.64

#1.7

2.8259

Carbonic anhydrase

Bovine I

- 19.7070

2.76

4.8476

Bovine II

- 19.7189

2.73

4.8686

Bovine/rabbit III

- 19.7020

2.78

4.8387

Rabbit IV

- 19.6876

2.82

4.8131

Canine/human V

- 19.6988

2.78

4.8330

Arithmetic mean

- 19.7030

2.77

4.8404

a Literature values.

bThe molecular sizes calculated are compared with the literature data and the percentage deviation indicated. Reproduced with permission from Rothe (1991).

Table 5 Marker proteinsthat can be used to estimate the native molecular size of proteins

Marker protein

Mr

Rs

Carbonic anhydrase

30 000

2.43

Ovalbumin

45000

3.05

Bovine serum albumin

67000

3.55

P-Galactosidase

116000

4.23

Lactate dehydrogenase

140000

4.20

Catalase

232000

5.25

Ferritin

440000

6.10

Thyroglobulin

669000

8.50

M (Da), Molecular mass; RS (nm), Stokes' radius of proteins. These markers can be taken when using PA gradients of 4-30% Tand a buffer of pH 8.4 (45 mmol L"1 Tris, 40 mmol L"1 boric acid, 1.25 mmol L"1 EDTA-Na2, pH 8.4).

M (Da), Molecular mass; RS (nm), Stokes' radius of proteins. These markers can be taken when using PA gradients of 4-30% Tand a buffer of pH 8.4 (45 mmol L"1 Tris, 40 mmol L"1 boric acid, 1.25 mmol L"1 EDTA-Na2, pH 8.4).

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