Amphipathic reagents

Amphipathic reagents have been used extensively to label biological membranes by Wisnieski and her group. A reagent in current use is 12-(4-azido-2-nitrophenoxy)stearoyl [I-l4C]glucosamine which can be made at a specific radioactivity of 50 mCi mmol-1 (Fig. 6.3). 

Many of the matters that were considered for simple hydrophobic reagents apply to amphipathic molecules and they will not be discussed again here. Like hydrophobic reagents, properly designed amphipathic reagents are convenient to use as they may be added directly to samples... 

However, since many of the traditional biotinylation reagents, such as NHS-LC-biotin contain hydrophobic spacers, their use with amphipathic liposomal constructions may not be entirely appropriate. A better choice may be to use a hydrophilic PEG-based biotin compound that creates a water-soluble biotin modification on the outer aqueous surface of the liposome bilayer. Biotinylation reagents of this type are discussed in Chapter 18, Section 3. 

Experiments in which the (l-subunil was labeled more heavily with amphipathic and phospholipid reagents have been described recently (Montecucco et al. 1981). 

The results of Fig. 14 suggested that the purification of the desired product would be best achieved by a multistep approach, i.e., an initial HILIC/CEC step followed by RPC of the desired peptide product. Another example of the resolving capability of the multistep HlLlC/CEC and RPC protocol is illustrated in Fig, 15, which outlines the purification of a synthetic 21-residue amphipathic a-helical peptide from the peptide crude mixture. From the RPC elution profile (top) of the crude peptide, a major peak is obtained with a number of more hydrophilic and hydrophobic impurities, some of which are eluted very close to the main component. Subsequent attempts to improve the isolation of this main peak by varying the pH and/or the ion-pairing reagent used in the RPC mobile phase were unsuccessful, thus ruling out the possibility of an efficient one-step purification by RPC, Application of mixed-mode HILIC/CEC to the crude mixture resulted in the middle elution profile, which shows a major peak (P) as well as several other smaller, yet substantial, peptide components. Collection of the major peak followed by RPC produced the bottom elution profile, with the... 

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