Paraffin oil protected high resolution hybrid isoelectric focusing for the demonstration of substitutions of neutral amino acids in denatured proteins: The case of four human transthyretin (prealbumin) variants associated with familial amyloidotic polyneuropathy
✍ Scribed by Prof. Dr. Klaus Altland; Pia Becher; Angelika Banzhoff
- Publisher
- John Wiley and Sons
- Year
- 1987
- Tongue
- English
- Weight
- 564 KB
- Volume
- 8
- Category
- Article
- ISSN
- 0173-0835
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✦ Synopsis
Paraffin oil protected high resolution hybrid isoelectric focusing for the demonstration of substitutions of neutral amino acids in denatured proteins: The case of four human transthyretin (prealbumin) variants associated with familial amyloidotic polyneuropathy
All four sequenced variants of human plasma transthyretin (TTR) with different substitutions of electrically neutral amino acids and associated with familial amyloidotic polyneuropathy (i. e. TTR(Met3'), TTR(Ala6'), T T R ( T Y ~~~) and TTR(SerS4)) have been separated from the normal TTR monomer by hybrid isoelectric focusing (HIEF) under denaturing conditions. The p1 differences from the normal monomer were between 0.0025 and 0.005 pH units corresponding to 0.7-1.4 % of a full charge unit difference. The detectability of these variants by HIEF under denaturing conditions (7 M urea, 50 mM dithiothreitol, 2 %Triton X-100) was explained assuming interaction ofthe amino acids at the substitution site with a charged amino acid in its close vicinity. It is proposed to distinguish a high from a low level of resolution for HIEF under denaturing conditions to underline the fact that the studied variants could only be detected by extreme flattening ofthe pH gradient. To provide protection against modification by the laboratory atmosphere the entire gel, including the electrode wicks and sample application site, was covered with alayer ofparaffin oil.