Glycoprotein Methods and Protocols - P7

Heterogeneity and Size Distribution of Gel-Forming Mucins 87

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From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins

Edited by: A. Corfield © Humana Press Inc., Totowa, NJ

Heterogeneity and Size Distribution

of Gel-Forming Mucins

John K. Sheehan and David J. Thornton

1. Introduction

The rheological properties of human mucus are dominated by the physical proper￾ties of large secreted O-linked glycoproteins often referred to as gel-forming mucins.

These molecules share the ability to assemble into long oligomeric structures via the

agency of disulfide bonds. There is evidence that at least four mucins—MUC2,

MUC5AC, MUC5B, and MUC6 (1–4)—are gel-forming mucins, and it is possible

that there are others. In isolating a mixture of mucins from mucus, there is consider￾able scope for heterogeneity in their mass and length owing to the presence of differ￾ent gene products that may themselves show polymorphism, different glycoforms of

the various gene products (see Chapter 7), and variable numbers of subunits contribut￾ing to the final polymer. It is possible that heterogeneity may be an important biologi￾cal property of gel-forming mucins and a key comparative characteristic when studying

the change of mucous properties through the course of disease.

This chapter describes only methods for assessing the heterogeneity of mucin

populations with regard to mass and size utilizing rate zonal centrifugation and electron

microscopy. Other methods for the absolute determination of molecular weight using

light scattering and analytical centrifugation are not described here because they require

expensive, specialized equipment and a detailed knowledge of their theoretical basis.

2. Materials

2.1. Extraction and Purification

See Chapter 1 for details.

2.2. Rate-Zonal Centrifugation

1. 6 M guanidinium chloride (GuHCl).

2. 8 M GuHCl.

3. Peristaltic pump.

4. Swing-out rotors and tubes (see Note 1).

5. Gradient maker (see Note 2).

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