MicroSolv Technology Corporation

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Chart
Peaks:
Between 5.8 and 6.5 minutes:
    2-sialo Tf
    3-sialo Tf
    4-sialo Tf
    5-sialo Tf
    6-sialo Tf

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printable Application Sheet

Separation of Transferrin Isoforms by CZE

Method:
Buffers and sample saturation solution from the CEoFix CDT™ kit were used to separate Bovine Holo Transferrin (Sigma T1283) with a purity about 95% of protein. Methodology used was supplied with the kit and no variations were made.

Discussion and Rationale:
Separation of glycol proteins and their isoforms is very important. Using this complete kit of premade buffers, it is easy to identify isoforms by Capillary Electrophoresis. Using the different isoforms as disease markers is a very important potential for this test.

Method Conditions:
Voltage: 30kV
Capillary: MicroSolvCE 50µm, Bare Fused Silica
Injection: 5 sec pressure
Run Buffer: CEoFix Accelerator (supplied) pH 8.5
Detection: UV 200nm
Sample: Holo Transferrin 9mg & 250µl BSA 50mg/ml & 50µl Fe3 Solution
Polarity: Normal
Temperature: 30°C
Pretreatment of Samples: Addition of Fe3 Solution (supplied), No incubation of serum

Background:

Transferrin is an iron-transport glycoprotein and consists of a polypeptide chain with two binding sites for iron and two N-linked oligosaccharide chains. The oligosaccharide chains are micro-heterogenous and carry sialic acid residues. Transferrin can be separated into several isoforms based on this structure. Asialo- and disialotransferrin, which are due to impaired glycosylation, are referred as carbohydrate-deficient transferrin (CDT). CDT is known as a valuable marker of chronic alcohol abuse. The main isotransferrin (tetrasialo) has a pI of 5.4 while the various isoforms, deficient in sialic acid, have a pI 5.6.

This capillary electrophoretic kit/method will determine the percentage of CDT in serum. You will be able to show a difference of migration between transferrin carrying two sialic acids after desialylation and the disialotransferrin from serum of alcoholic patients. Also, the transferrin carrying three sialic acids after desialylation and the trisialotransferrin co-migrate. This suggests that the disialotransferrin fraction not only carries less sialic acid, but also lacks one of the entire carbohydrate chain.

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