Covalent chromatography

The most useful technique would be affinity chromatography. Covalently attach the purified hormone to sepharose or some other form of an insoluble bead (see problem 4), and use the hormone-bead complex to fill a column. Homogenize liver cells, and add the homogenate to the column. Receptors should stick on the column because their hormone-binding sites are complementary in shape to the covalently bound hormone. You could elute the receptors from the column as hormone-receptor complexes by adding free hormone to compete with the hormone that is bound to the column. 

Chemiselective adsorption is the process where the affinity is based on chemically defined nonbiological interactions. It includes hydrophobic chromatography, ion-exchange chromatography, covalent chromatography (active thiols, Hg +, etc), and borate complexes. 

Covalent chromatography Covalent binding Functional groups... 

Purified by (NH4)2S04 fractionation, followed by PC cellulose chromatography and affinity chromatography (using Sepharose 4B to which (G)n was covalently bonded). [Schmukler et al. J Biol Chem 250 2206 7975.]... 

Synthetic chiral adsorbents are usually prepared by tethering a chiral molecule to a silica surface. The attachment to the silica is through alkylsiloxy bonds. A study which demonstrates the technique reports the resolution of a number of aromatic compoimds on a 1- to 8-g scale. The adsorbent is a silica that has been derivatized with a chiral reagent. Specifically, hydroxyl groups on the silica surface are covalently boimd to a derivative of f -phenylglycine. A medium-pressure chromatography apparatus is used. The racemic mixture is passed through the column, and, when resolution is successful, the separated enantiomers are isolated as completely resolved fiactions. Scheme 2.5 shows some other examples of chiral stationary phases. 

Shanzer and Mayer-Shochet have utilized disubstituted stannoxanes as covalent templates for the formation of tetralactones. In this procedure, a dialkyl stannoxane is condensed with a glycol to form a cyclic-distannoxane as illustrated below. In a typical preparation, the distannoxane (i) was dissolved in CCI4 (ca. 0.04 At) at reflux and sebacoyl chloride (2 eq) was added dropwise at reflux. After heating for 20 h, the solution was concentrated to a solid. Chromatography afforded the tetralactone in 30% yield. ... 

PTC was coupled with (Pro-Ala-Gly)n, n = 12. The covalently bridged three-chain compound could be separated by gel chromatography from the respective two chains and the single chain in low yield, PTC-[(Pro-Ala-Gly-)12]3 shows a strong fold (Figs. 20,21) in comparison to the dimer or the monomer. 

Candidates for the renal brush border Na /H exchanger transport protein identified by covalent labeling, affinity chromatography, or other methods... 

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