Hydrophobic reagents

SIAX is a hydrophobic reagent that should penetrate membrane structures with good efficiency. The crosslinker must be solubilized in organic solvent (DMF or DMSO) prior to transferring a small amount to an aqueous reaction medium. 

At this point, the carrier protein was released from a suspension of the membranes by addition of the hydrophobic reagent octylglucoside in the presence of E. coli phos-... 

The isolation of a stable P-450 from membranes by Horie and Kinoshita (22), Appleby (3), and Gunsalus et al. (6) has revealed the nature of this pigment. The optical absorption characteristics are given in Table 2 for comparison with Table 1. The isolated P-450 shows all of the properties of the membrane bound compound including the sensitivity to hydrophobic reagents and mercurials. 

Some miscellaneous methods which may grow in importance deserve a brief review. The potential use of photogenerated reagents in experiments where time-dependent phenomena are examined was mentioned in Chapter 1. Many methods for investigating rapid events in biological systems, such as the use of fluorescent probes, yield little structural information. Examples of experiments with photoactivatable reagents that do yield such information have appeared recently. They include the use of a hydrophobic reagent to monitor the interaction of cholera toxin with membranes... 

In recent years there has been considerable interest in the structure of biological membranes, and photochemical reagents capable of yielding low resolution structural information about membrane proteins have been developed. Several examples of photochemical surface-labeling reagents have appeared, and much effort has been devoted to the development of photoactivatable hydrophobic reagents for labeling from within the lipid bilayer. 

Photoactivatable, hydrophobic reagents for membranes were developed during the same period as photoactivatable, surface labeling reagents, but... 

A simple hydrophobic reagent is a small radiolabeled organic molecule that can be photochemically activated after it has bound to a lipid bilayer. The major merit of such reagents in comparison to phospholipid analogs is that they are simple to use with native membranes. Klip and Gitler (1974)... 

It should be pointed out that despite their high reactivity carbenes are selective, and while they will react with hydrocarbons in the absence of nucleophiles, a nucleophilic group exposed within the bilayer will preferentially be attacked. Some properties of the three most thoroughly tested reagents iodonaphthyl azide, adamantane diazirine, and 3-trifluoromethyl-3-(m-iodophenyl)diazirine are summarized in Table 6.1. There follows a brief description of a typical experimental procedure, a discussion of control experiments, and several caveats, followed by some examples of the applications of hydrophobic reagents. 

Several control experiments have been suggested to confirm that hydrophobic reagents do label from within the lipid bilayer (Bercovici et al. 1978 Bayley and Knowles, 1980). Even though the reagents listed in Table 6.1 have been thoroughly tested and their virtues and limitations described, the wise investigator will repeat at least some of the controls described here with his system. 

Some examples of labeling with hydrophobic reagents... 

Both [5-12SI]iodonaphthylazide (Karlish et al. 1977) and [3H]adaman-tane diazirine (Farley et al. 1980) labeled the a-subunit strongly and the (3-subunit hardly at all. The simplest interpretation of this result is that the (3-subunit is a peripheral protein, tightly attached to the a-subunit. The possibility remains, however, that the (3-subunit is relatively unreactive towards hydrophobic reagents even though the two used in these studies have quite different properties. A further possibility that must be taken into account when considering multisubunit membrane proteins is that (3 penetrates the bi layer but is shielded from the hydrocarbon phase by neighboring a-subunits. ... 

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... 

The looping around of photoactivated fatty acyl chains to the membrane surface, with subsequent reaction there or at an intervening point, is a distinct possibility (Bayley and Knowles, 1978a). Such events would be analogous to the exchange of simple hydrophobic reagents into the aqueous phase resulting in the derivatization of peripheral proteins, but in the case... 

Special starch products have been developed for use as thickeners in coatings that are based on synthetic latex or resin binders. Starch-based thickeners can have associative properties when substituted with a hydrophobic reagent. They provide an alternative for cellulose-based thickeners, but their use is limited. 

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