REVERSE-PHASE HPLC OF THE HYDROPHOBIC PULMONARY SURFACTANT PROTEINS -DETECTION OF A SURFACTANT PROTEIN-C ISOFORM CONTAINING N-EPSILON-PALMITOYL-LYSINE

A reverse-phase HPLC protocol for analysis of strictly hydrophobic pep tides 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 pu lmonary surfactant-associated proteins B (SP-B) and C (SP-C) with this method reveals several features. (I) SP-B and SP-C retain their secon dary structures and separate by about 15 min over a 40 min gradient. S P-B is more hydrophilic than SP-C, which in turn behaves chromatograph ically like palmitoyl-ethyl ester. (2) SP-C exhibits isoforms addition al to the major form characterized previously, which contains two thio ester-linked palmitoyl groups. The isoforms now observed contain one o r three palmitoyl moieties and constitute together 15-20% of the major form. The tripalmitoylated species contains a palmitoyl group linked to the E-amino group of Lys-11, as concluded from the elution position , MS and amino acid sequence analysis. The tripalmitoylated form incre ases relative to the dipalmitoylated form on incubation of SPC in a ph ospholipid environment. An N-epsilon-bound palmitoyl moiety constitute s a third mode of fatty acyl modification of proteins, in addition to the established N-alpha-bound myristoyl groups and S-bound palmitoyl c hains. (3) The dimeric structure of SP-B, lacking covalent modificatio ns, is confirmed by MS detection of the dimer. No SP-B isoforms were d etected. (4) Denatured, nonhelical SP-C can be distinguished chromatog raphically from the native alpha-helical peptide. (5) HPLC of SP-C at 60-75 degrees C reveals an isoform containing an extra 14 Da moiety co mpared 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 bef ore functional studies. It might also prove useful for analysis of oth er strictly hydrophobic polypeptides.