Abstract
This chapter discusses the application of high-performance liquid chromatography (HPLC) methods to the separation of peptide mixtures from the L12 homolog from the large subunit of M. vannielii ribosomes. Further, different HPLC techniques—namely, size exclusion, reversed-phase, and ion-exchange chromatography—are discussed for their ability to resolve different archaebacterial r-protein mixtures. For the identification of proteins after HPLC fractionation, a gel system suitable for archaebacterial protein mixtures was miniaturized so that particular proteins could easily be identified using only small aliquots of the peaks (nanogram amounts) to conserve valuable materials for other purposes. Yields of proteins were calculated by amino acid analysis using precolumn derivatization with o -phthaldialdehyde and reversed phase HPLC. Using the HPLC methods for archaebacterial protein and peptide separations described in this chapter, it is possible to arrive at new sequence data of proteins that would not be feasible without the advanced methodology. The applied methods are suitable not only to structural investigations on r-proteins but also to studies of other proteins of limited sources— for example, multienzyme complexes, receptor proteins, or protein constituents of other cell organdies.
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