Estimation of Molecular Mass of Denatured Proteins and Small Peptides

When sDs electrophoresis is performed in a linear PA gradient gel of 3-30% T, a linear relationship can be set up between the logarithm of the mol mass (log Mr) and the log of the PA concentration (log T) reached by proteins after a certain time of electrophoresis. The validity of the corresponding relationship log Mr = —ax log T + b has been confirmed with some 40 proteins between 14 and 950 kDa. In PA gradient gels in the presence of sDs the molar mass of both unreduced and 2-mercaptoethanol-reduced proteins as well as the molar mass of glycoproteins can be determined with the same accuracy ( ± 5%, Table 12). Ribonuclease and lysozyme binding normal amounts of sDs migrate anomalously in homogeneous sDs gels but not in sDs PA gradient gels. Papain and pepsin, which also bind only traces of SDS, migrate regularly in SDS PA gradient gels.

The migration distance of proteins in linear sDs PA gradient gels and their respective mol mass can also be correlated by the equation:

where D (mm) is the migration distance. This relationship can be applied to SDS-complexed and reduced and to SDS-complexed nonreduced proteins, to glycoproteins and to carbohydrate-free proteins (Figure 15). The relationship is not affected by the buffer system, the concentration of the cross-linker within 1-8% C or the concentration range of the gradient within 3-30% T at the commonly used gel length of 8-15 cm. The value of the constants a and b, on the other hand, are changed when the experimental parameters are altered. If SDS electrophoresis is performed in a linear gradient gel of approximately 6-27% T, the relationship log Mr = — ax^D + b is practically independent of the time of electrophoresis. This means that the molecular mass estimation can be made when the best resolution of a set of proteins has been obtained. It is not necessary to wait until the proteins have reached their exclusion pore size. On the contrary, under prolonged electrophoresis protein-SDS complexes can reach a pore size where the complexing SDS is stripped off the protein molecules which leads to erroneous banding patterns. This is particularly

Table 11 Gel and buffer systems used in SDS PA gradient gel electrophoresis to separate denatured proteins

PA range (% T)

Gel shape

Buffer systems

Current or Running

Correlation

Notes

Authors

(acrylamide-Bis)

(dimensions

voltage

time (h)

(Mr range

(mm))

Gel buffer

Electrode buffer

per gel

(kDa))

3-30

Column

0.1 mol L~1

0.1 mol L~1

4 mA

24

_

a

Exposito and

(30 : 0.8)

(150x6)

Na-phosphate,

Na-phosphate,

(12-125)

Obijeski

0.1% SDS,

0.1% SDS, pH

(1976)

5-15% (v/v)

7.0

glycerol, pH 7.0

3-30

Slab gel

10.75 g Tris,

0.01 mol L~1

40 V

16

log Mr vs.

b

Lambin etal.

(9.62 : 0.38)

(length: 80)

5.04g boric acid

Na-phosphate,

log T

(1976),

0.93 g

1 % SDS, 1 % 2-

(13-950)

Lambin

EDTA-Na2,

mercaptoethanol,

(1978)

pH 7.2

pH 7.2

1.5-40

Microcolumn

0.1 mol L~1

29 g glycine plus

60 V

2

log Mr vs.

c

Ruchel etal.

(12.57: 1)

i.d. 0.43,

Na-phosphate,

Tristo pH 8.4, 1 g

Rf

(1974)

length 15

pH 7.2,

SDS,

(13-300)

0.1% SDS or

H2O to 1000 mL

0.35 mol L~1

Tris-sulfate, 0.1 %

SDS, pH 8.5, or

0.05 mol L~1

Tris-glycine,

0.1% SDS, pH

8.4, or

0.065 mol L~1

Tris-borate, 0.1 %

SDS, pH 9.3

1.5-40

Microcolumn

4 g Tris and

29 g glycine plus

60 V

0.33

log Mr vs.

c

Ru chel etal.

(12.57: 1)

(i.d. 0.43,

H2SO4 to pH 8.4,

Tristo pH 8.4, 1 g

Rf (13-300)

(1974)

length 15)

H2O to 10 mL

SDS, H2O to

1000 mL

3-30

Slab gel

0.04 mol L~1 Tris,

Same as gel

150 V

0.5-8

log Mr vs.

d

Rothe (1982)

(28 : 1)

(width 80,

0.02 mol L~1

buffer

JO (13-950)

length 80,

Na-acetate,

thickness 1)

0.02 mol L~1

Na-EDTA,pH

7.4, 0.2% SDS

aGels were stored at room temperature before use in a solution which contained 0.1 mol L~1 Na-phosphate, 0.01% SDS, 15% glycerol, 2mmolL~1 EDTA-Na2 and 0.01% NaN3. Samples were dissolved at 100°C for 3 min in 0.01 phosphate buffer, pH 7, containing 2.5% SDS, 5% 2-mercaptoethanol, 10% glycerol and 0.005% Bromophenol blue. On each column 20-100 ^g protein was loaded.

b T%) g acrylamide plus g Bis per 100 mL solvent. Protein samples (0.5 mg mL~1) were incubated in 0.01 mol L~1 phosphate buffer, containing 1 % SDS, pH 7.2 for 3 min in a 100°C bath; for cleavage of disulfide bridges 1 % 2-mercaptoethanol was added. The % Tconcentration reached by each protein after electrophoresis was determined and log Tplotted versus log mol mass. ^Resolution was found to be better in discontinuous than in continuous buffer systems. Samples (1 mg protein mL~1) were treated for 2 min at 100°C with 1 % SDS and 1 % 2-mercaptoethanol in 0.035 mol L~1 Tris-sulfate, pH 8.6, 0.35 mol L~1 Tris-sulfate, pH 8.6 or 0.1 mol L~1 phosphate. Complete removal of SDS from proteins can be achieved with SDS-free electrode buffers. The activity of ß-galactosidase denatured with SDS and separated on an SDS-free PA gradient gel could be restored to 10%. djD, square root of migration distance (D (mm)). Re-evaluation of the data from Lambin (1978), Lasky (1978) and Poduslo and Rodbard (1980) confirmed the validity of the log Mr - J D relationship, found when evaluating time-dependent SDS-porosity gradient gel electrophoresis using marker proteins in the range of 10-330 kDa.

References as given in Rothe and Maurer (1986). Reproduced with permission from Rothe and Maurer (1986).

true when in an alkaline buffer system the upper electrode buffer contains no SDS.

In SDS electrophoresis with linear PA gradients ranging from 3 to 30% T, polypeptides in the range of 1.4-10 kDa cannot be resolved. Separation is possible, however, in 10-18% T gels in the presence of 0.1% SDS and 7molL_1 urea (cf. Tables 13 and 14).

Table 12 Separation characteristics of some proteins in SDS PA gel electrophoresis and deviation of calculated mol masses from those given in the literature

No. Protein Mr (Da) 3-30% T C = 8.4%a 3-30% T, C = 3.8%b

(literature value) £ MF T M^ D M" T M (mm) $ % (%) ± % (mm) $ % (%) $ %

1

Prealbumin

13 745

51.5

-0.4

20.7

- 4.0

56

- 1.7

22.4

- 6.8

2

Lysozyme

14314

53.5

- 16.5

21.4

- 20.4

51.5

# 16.9

20.8

# 13.6

3

Ribonuclease B

14 700

52

- 10.0

20.9

- 14.0

55.5

- 6.0

22.2

- 10.3

4

Haemoglobin

15 500

51

-8.6

20.5

- 11.2

55

- 8.7

22

- 12.3

5

Avidin

16 000

49.5

- 1.7

20

- 4.2

51

# 7.1

20.6

# 4.8

6

Soybean trypsin inhibitor

20095

47

-6.6

19.2

-9.2

50

# 10.4

20.3

- 12.7

7

Papain

23426

44.5

- 4.0

18.3

- 4.8

48

# 15.1

19.6

- 16.5

8

a-chain of IgG

23500

9

Chymotrypsinogen A

25666

43.5

-5.6

18

-7.0

44

# 4.8

18.2

- 4.8

10

Carbonic anhydrase B

28739

41

#1.8

17.1

#2.2

42

# 5.5

17.5

- 4.7

11

Carboxypeptidase A

34409

40

-8.1

16.7

-6.3

39.5

- 9.5

16.7

- 9.0

12

Pepsin

34700

37.5

#11.0

15.9

# 12.5

37.5

# 0.4

16

#1.8

13

Glycerol-3-phosphate dehydrogenase

35700

37.5

#7.9

15.9

#9.4

36

# 6.4

15.5

# 7.9

14

Lactate dehydrogenase

36180

37.5

#6.4

15.9

#7.9

37

- 1.0

15.8

#1.0

15

Aldolase

38994

36

#11.5

15.4

# 13.1

35

# 3.2

15.1

# 6.0

16

Alcohol dehydrogenase

39805

35.5

#13.8

15.2

# 16.4

34.5

# 4.2

14.9

# 7.7

17

arAcid glycoprotein

40000

35

# 18.0

15

# 21.7

32

# 20.6

14.1

# 23.9

18

Ovalbumin

43000

35.5

#5.4

15.2

#7.8

35.5

- 9.1

15.3

- 7.2

19

Fibrinogen y chain

47000

20

Glutamate oxalacetate transaminase

50000

32.5

# 16.7

14.2

# 19.2

29

# 16.6

13

# 21.9

21

Heavy chain IgG

50000

22

Fibrinogen ß chain

56000

23

Catalase

57500

32

#5.9

14

#9.1

29

#1.4

13

# 6.0

24

Fibrinogen a chain

63500

25

Albumin monomer

66290

31.5

# 10.7

13.8

# 14.9

28

# 8.2

12.7

- 12.3

26

Heavy chain IgM

72000

27

Transferrin

76000

30.5

-8.6

13.5

-6.0

24

# 7.6

11.3

# 12.4

28

Plasminogen

81 000

29

- 1.8

13

#0.7

23

# 8.5

11

# 12.2

29

Phosphorylase b

96800

25.5

# 14.2

11.8

# 17.1

21

# 5.3

10.3

# 8.9

30

Ceruloplasmin

124000

23.5

#8.8

11.1

#11.6

17

# 13.2

8.9

# 16.2

31

Albumin, dimer

132580

24

-3.3

11.2

#1.4

17.5

#1.6

9

# 6.2

32

Immunoglobulin G

150000

21.5

# 10.6

10.4

# 13.4

15

#11.4

8.2

# 13.4

33

Immunoglobulin A

160000

22

#1.6

10.6

#0.1

14

# 14.5

7.8

# 17.2

34

Reduced a2-macroglobulin

190000

35

Albumin, trimer

198870

19.75

#0.8

9.8

#2.6

12

#11.8

7.1

# 12.6

36

Immunoglobulin A

320000

16

-3.1

8.5

-3.5

9

- 4.0

6.1

- 8.4

37

Thyroglobulin

330000

16.5

- 11.6

8.7

- 12.4

11

- 25.4

6.8

- 26.6

38

Fibrinogen

340000

15

#3.3

8.2

#0.4

8.5

- 4.2

5.9

- 8.8

39

a2-Macroglobulin

380000

15.5

- 13.2

8.3

- 13.1

8

- 9.0

5.8

- 16.0

40

Immunoglobulin A, trimer

480000

13

- 4.8

7.5

-9.5

6.5

- 12.4

5.2

- 20.6

41

Immunoglobulin M Average % deviation Lambin (1978)

950000

8.75

-9.1 ±7.8

6

- 20.1 ±9.6 ±5.9

± 8.7

±11.2 ± 7.4

aThe gel buffer contained no 2-mercaptoethanol (gel length 78.5 mm).

bGel buffer with 2-mercaptoethanol (gel length 81 mm) , gel and electrode buffer as well as conditions of electrophoresis as given in Table 8. Mr (Da) , mol mass; D (mm) , migration distance; T(%) , g acrylamide plus g Bis per 100 mL , as reached by a protein. cMr $ %, %-deviation of calculated mol mass from the literature value using the relationship log Mr = ax^D + b. dMr $ %, %-deviation of calculated mol mass from the literature value using the relationship log Mr = ax^T+ b. Reproduced with permission from Rothe and Maurer (1986).

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