Purified Bovine Neurofilament NF-H (Nfh, NEFH) Prot-m-NF-H

      Neurofilaments are the 10 nm or intermediate filament proteins found specifically in neurons, are composed predominantly of four major proteins called NF-L, NF-M, NF-H and α-internexin (1). NF-H is the neurofilament heavy or high molecular weight polypeptide and runs on SDS-PAGE gels at 200-220kDa in the heavily phosphorylated axonal form. The molecule has an unusual and interesting region consisting of multiple Lysine-Serine-Proline peptides, about 40 of these in human. These peptide repeats are heavily phosphorylated on the Serine residues in axons. Enzymatic removal of these phosphate groups will increase the SDS-PAGE mobility to about 160kDa, likely due to protein conformational changes due to the removal of charge (2). Even the non-phosphorylated form runs aberrantly on SDS-PAGE, as the real molecular weight of NF-H is about 110kDa, with some variation in different species. This is likely due to an unusually high content of charged amino acids. Non-phosphorylated forms of NF-H are found in dendrites and perikarya and early in development, but the majority of NF-H in the adult is this heavily phosphorylated axonal form.

This protein was purified using a modification of the method of Leung and Liem (3). This involves homogenization followed by pelleting of axonal material. The axonal material is then extracted with 1% Triton X-100 to remove myelin by centrifugation several times. The pellet is then dissolved in 6M urea buffer to dissolve cytoskeletal material. This material was then mixed with hydroxyapatite which was then washed with 10mM Urea plus 10mM phosphate buffer pH=7.0. The NF-H protein does not binds to hydroxyapatite under these conditions but can eluted, along with NF-L and NF-M, by washing with 300mM phosphate in 6M urea. NF-H is purified away from NF-L and NF-H using ion exchange chromatography on a DEAE column.

1. Perrot, R., Berges, R., Bocquet, A and Eyer, J. Review of the multiple aspects of neurofilament functions, and their possible contribution to neurodegeneration. Mol. Neurobiol 38:27-65 (2008).
2. Julien, J-P. and Mushynski, W. E. Multiple phosphorylation sites in mammalian neurofilament polypeptides. J. Biol. Chem. 257:10467-10470 (1982).
3. Leung, C. L. and Liem, R. K. H. Isolation of intermediate filaments. Prot. Cell Biol. 3:Unit 3.23 doi: 10.1002/0471143030.cb0323s31 (2006).