The application of reversed-phase chromatography to peptide separations has revolutionized peptide chemistry. Single amino acid substitution is usually detectable in peptides containing less than 20 amino acids. However, it is worth noting that there may be broad differences in the way various reversed-phase packings separate polypeptides. These differences become particularly prominent in the higher molecular weight hydrophobic peptides.
Recently, it was demonstrated that pore diameter influences the resolution of reversed-phase columns'J. Through the use of supports of 300 and 500 A pore diameter it was shown that the resolution of high-molecular-weight collagens could be enhanced over that obtainable with a support of 100 8, pore-diameter'. In view of the increasing value of wide-pore supports in reversed-phase liquid chromatography of biopolymers3 and of our interest in the characterization of the light chains associated with myosin from various types of tissues, we decided to adapt this method to the separation of myosin light chains from fast and slow muscles of the rat. These proteins are characterized by a molecular weight ranging from 27,000 to 16,000 daltons. In particular, light chains (LC) of fast (F) muscles are LClF, LC2F and LC3F (25,000; 18,000 and 16,000 daltons, respectively), while the light chains of slow (S) muscles are LClS and LC2S (27,000 and 25,000 daltons, respectively)4F5.
In this paper it will be shown that these proteins are separated in less than 30 min using a wide-pore (330 A) reversed-phase column. The results also show that reversed-phase liquid chromatography makes possible the resolution of homologous polypeptides characterized by a similar molecular weight in a submilligram sample and greatly facilitates the analysis of biopolymers available in only small amounts.
EXPERIMENTAL
Reagents
Acetonitrile (HPLC grade) and trifluoroacetic acid (TFA) were obtained from either BDH Chemicals (Poole, U.K.) or Baker (Deventer, The Netherlands). Water was glass-distilled and filtered through a 0.45~pm Millipore filter. Mobile phase solutions were degassed by sonication before use.