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Reverse-phase HPLC ...
Reverse-phase HPLC of the hydrophobic pulmonary surfactant proteins: detection of a surfactant protein C isoform containing Nepsilon-palmitoyl-lysine
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Gustafsson, M (författare)
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- Curstedt, T (författare)
- Karolinska Institutet
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- Jornvall, H (författare)
- Karolinska Institutet
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- Johansson, J (författare)
- Karolinska Institutet
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(creator_code:org_t)
- Portland Press Ltd. 1997
- 1997
- Engelska.
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 326326 ( Pt 3), s. 799-806
- Relaterad länk:
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https://europepmc.or...
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http://kipublication...
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https://doi.org/10.1...
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Abstract
Ämnesord
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- A reverse-phase HPLC protocol for analysis of strictly hydrophobic peptides and proteins was developed. Peptide aggregation is minimized by using only 25–40% water in methanol or ethanol as initial solvents and subsequent elution with a gradient of propan-2-ol. Analysis of the pulmonary surfactant-associated proteins B (SP-B) and C (SP-C) with this method reveals several features. (1) SP-B and SP-C retain their secondary structures and separate by about 15 min over a 40 min gradient. SP-B is more hydrophilic than SP-C, which in turn behaves chromatographically like palmitoyl-ethyl ester. (2) SP-C exhibits isoforms additional to the major form characterized previously, which contains two thioester-linked palmitoyl groups. The isoforms now observed contain one or three palmitoyl moieties and constitute together 15–20% of the major form. The tripalmitoylated species contains a palmitoyl group linked to the ϵ-amino group of Lys-11, as concluded from the elution position, MS and amino acid sequence analysis. The tripalmitoylated form increases relative to the dipalmitoylated form on incubation of SP-C in a phospholipid environment. An Nϵ-bound palmitoyl moiety constitutes a third mode of fatty acyl modification of proteins, in addition to the established Nα-bound myristoyl groups and S-bound palmitoyl chains. (3) The dimeric structure of SP-B, lacking covalent modifications, is confirmed by MS detection of the dimer. No SP-B isoforms were detected. (4) Denatured, non-helical SP-C can be distinguished chromatographically from the native α-helical peptide. (5) HPLC of SP-C at 60–75 °C reveals an isoform containing an extra 14 Da moiety compared with the main form. This is concluded to arise from inadvertent methyl esterification of the C-terminal carboxy group. In conclusion, this HPLC method affords a sensitive means of assessing modifications and conformations of SP-B or SP-C in different disease states and before functional studies. It might also prove useful for analysis of other strictly hydrophobic polypeptides.
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