Proteins affinity HPLC

Employ sequential separation processes based on different physical, chemical, or biochemical properties that are synergistic, and thus orthogonal, rather than repetitive, i.e., use a metal ion-affinity HPLC procedure prior to a size-exclusion step, a hydrophobic interaction HPLC step after an ion-exchange HPLC procedure, a biomimetic HPLC step before a biospecific affinity HPLC step, etc. This rule is in accord with the orthogonality rule, which anticipates that the most efficient separation procedures are ones that take advantage of the anisotropy of molecular physicochemical properties of the target protein or polypeptide rather than the commonality of the molecular features. 

Table 8. (i) Commercially available columns for affinity HPLC of proteins. 

Mifepristone. After oral adininistration, peak plasma levels of mifepristone (84) (RU 486) are reached in 1 h and over 95% was bound to plasma proteins (351,352). The plasma half-life of RU 486 is approximately 24 h (352,353). In humans, monodemethylated (98), didemethylated (99) and alcohoHc nondemethylated (100) metaboHtes of RU 486 have been identified (351). These metaboHtes show some progestin-binding affinity, approximately five to ten times lower than that of RU 486 itself. RU 486 and its metaboHtes can be measured by radioimmunoassay and hplc (353,354). 

The application of monoliths for the analysis of proteins by CEC has been demonstrated by several workers. In these investigations the various mechanisms of interactions such as affinity, ion-exchange, and hydrophobic interactions have been demonstated. " Again it has been demonstrated that CEC produces shorter separation times and that a smaller amount of sample is required compared to conventional HPLC. 

Of course, the goal of every synthetic organic chemist is to obtain crystalline products, and a few cases of crystalline synthetic proteins have been reported. These include the ribonuclease A synthesized in solution/35 which crystallized after chemical workup and affinity chromatography (see Section 5.1.6.2.2). Further examples include an HIV protease analogue/701 a ubiquitin analogue/59 and monellin/89 which were each prepared by solid-phase methods and purified by HPLC. 

Ellman s reagent has been used not only for the determination of sulfhydryls in proteins and other molecules, but also as a precolumn derivatization reagent for the separation of thiol compounds by HPLC (Kuwata et al., 1982), in the study of thiol-dependent enzymes (Masamune etal., 1989 Tsukamoto and Wakil, 1988 Alvear et al., 1989), and to create sulfhydryl-reactive chromatography supports for the coupling of affinity ligands (Jayabaskaran etal., 1987). Another important use of the compound... 

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