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APPLICATION NOTE

AN010402-22

Separation of Cytochrome c Isoform on STYROSä SE: Strong cation exchanger.

 

The characterization of closely related forms a proteins, known as “isoform”, is essential as they may indicate:

a-       Specific structural change during the purification process or storage.

b-       Post-translational changes such as glycosylation, phosphorylation or lipidation.

c-       Substitution of one or more amino acids as a result of gene mutation.

Since the isoelectric point (pI) of protein isoforms are very close, it is important to use very efficient HPLC columns to properly separate them.

Ion exchange chromatography with STYROS™ columns has been very effective in achieving high level of separations.

The following examples show:

1-       the separation of 2 close isoform of Cytochrome c from bovine and horse heart (figure 1)

2-       the separation of normal hemoglobin A from the abnormal form S (figure 2).

The column is a strong cation exchanger STYROS™ SE with medium capacity.

 

 Figure 1
STYROS™SE/XP, Strong Cation Exchanger
4.6 x 250 mm. 0.5 ml/min (180 cm/hr), at pH= 7

  

 

Table 1. Operating Parameters.

HPLC System.

HP 1100

Columns

STYROS™  SE/XP 4.6 x 250 mm

Mobile Phase

A: 20 mM Bis-Tris-HCl, pH = 7 and 6
B: A + 1 M NaCl, Ph=7 and 6

Flow rates

As indicated

Gradient

8 to 17 % B in 9 cv for the separation of Cytochrome c,
4 to 30 % B in 11 cv for the separation of Hemoglobin.

Temperature

30°C

Detection

280 nm

Injection volume

25 ml

Samples

Cytochrome c from horse and bovine heart (5 mg/ml each).
Hemoglobin A and S (5:1.5 mg/ml)

 

 

Figure 2
 STYROS™SE/XP. Strong Cation Exchanger
4.6 x 250 mm. 5 ml/min (1,800 cm/hr), at pH=6

 

  

Hb S differs from Hb A by only one amino acid in position 6 of the b-chain: the substitution of Glutamic acid by Valine. The a-chain of the a2 b2 heterotetramere are similar.

The Cytochrome C variants chosen here differ only by 3 amino acids, yet their separation was performed at linear flow rates of 180 cm/hr.

Notice the linear velocity of 1,800 cm/hr (based on an empty column), far exceeds those used with either soft gel or non pervious media.
 

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