High-performance liquid chromatography and photoaffinity crosslinking to explore the binding environment of nevirapine to reverse transcriptase of human immunodeficiency virus
| Autor: | D E, Palladino, J L, Hopkins, R H, Ingraham, T C, Warren, S R, Kapadia, G J, Van Moffaert, P M, Grob, J M, Stevenson, K A, Cohen |
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| Rok vydání: | 1994 |
| Předmět: |
Benzodiazepinones
Photochemistry Pyridines Affinity Labels RNA-Directed DNA Polymerase Azepines Peptide Mapping HIV Reverse Transcriptase Cross-Linking Reagents HIV-1 Humans Reverse Transcriptase Inhibitors Spectrophotometry Ultraviolet Trypsin Nevirapine Sequence Analysis Chromatography High Pressure Liquid |
| Zdroj: | Journal of chromatography. A. 676(1) |
| ISSN: | 0021-9673 |
| Popis: | Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activity of reverse transcriptase of human immunodeficiency virus type-1. Nevirapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 181 and 188 of the p66 subunit of reverse transcriptase. The chromatography which was utilized to explore this binding site is described. BI-RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of nevirapine, are each crosslinked to reverse transcriptase. The use of several HPLC-based techniques employing different modes of detection makes it possible to demonstrate a dramatic difference between the two azido analogues in crosslinking behavior. In particular, by comparing HPLC tryptic peptide maps of the photoadducts formed between reverse transcriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and hence exploitable for the identification of the specifically bonded amino acid residue(s). In addition, comparison of the tryptic maps also makes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectroscopy to identify the crosslinked amino acid residue. |
| Databáze: | OpenAIRE |
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