High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde

Coordinate induction of phase 2 proteins and elevation of glutathione protect cells against the toxic and carcinogenic effects of electrophiles and oxidants. All inducers react covalently with thiols at rates that are closely related to their potencies. Inducers disrupt the cytoplasmic complex between the actin-bound protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the antioxidant response element (ARE) of phase 2 genes and accelerates their transcription. We cloned, overexpressed, and purified murine Keap1 and demonstrated on native gels the formation of complexes of Keap1 with the Neh2 domain of Nrf2 and their concentration-dependent disruption by inducers such as sulforaphane and bis(2-hydroxybenzylidene)acetone. The kinetics, stoichiometry, and order of reactivities of the most reactive of the 25 cysteine thiol groups of Keap1 have been determined by tritium incorporation from [3H]dexamethasone mesylate (an inducer and irreversible modifier of thiols) and by UV spectroscopy with sulforaphane, 2,2′-dipyridyl disulfide and 4,4′-dipyridyl disulfide (titrants of thiol groups), and two closely related Michael reaction acceptors [bis(2- and 4-hydroxybenzylidene)acetones] that differ 100-fold in inducer potency and the UV spectra of which are bleached by thiol addition. With large excesses of these reagents nearly all thiols of Keap1 react, but sequential reaction with three successive single equivalents (per cysteine residue) of dipyridyl disulfides revealed excellent agreement with pseudo-first order kinetics, rapid successive declines in reaction velocity, and the stoichiometric formation of two equivalents of thiopyridone per reacted cysteine. This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages. The most reactive residues of Keap1 (C257, C273, C288, and C297) were identified by mapping the dexamethasone-modified cysteines by mass spectrometry of tryptic peptides. These residues are located in the intervening region between BTB and Kelch repeat domains of Keap1 and probably are the direct sensors of inducers of the phase 2 system.

https://www.pnas.org/content/pnas/99/18/11908.full.pdf

Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants

Spectrophotometric Assay Using o-Phthaldialdehyde for Determination of Proteolysis in Milk and Isolated Milk Proteins

Signalling by steroid hormones is mediated by receptor proteins that bind hormonal ligands and regulate the transcription of specific genes. The heat-shock protein hsp90 seems to associate selectively with unliganded receptors (aporeceptors), but it has not been determined whether this interaction affects receptor function in vivo. To address the role of hsp90, we have taken advantage of the capacity of mammalian steroid receptors to function in yeast. We constructed a strain of Saccharomyces cerevisiae in which hsp90 expression was regulatable and could be reduced more than 20-fold relative to wild type. At low levels of hsp90, aporeceptors seem to be mostly hsp90-free, yet fail to enhance transcription; on hormone addition, the receptors are activated but with markedly reduced efficiency. Thus hsp90 does not inhibit receptor function solely by steric interference; rather, hsp90 seems to facilitate the subsequent response of the aporeceptor to the hormonal signal. This is the first biological evidence that hsp90 acts in the signal transduction pathway for steroid receptors.

Reduced levels of hsp90 compromise steroid receptor action in vivo

Primary amino acids react with o-phthaldialdehyde in the presence of mercaptans to form intensely fluorescent derivatives. By the use of reverse-phase high-performance liquid chromatography, a mixture containing 26 of these derivatives was efficiently resolved with an analysis time of less than 35 minutes. The quantitation of the individual amino acids was reproducible with an average relative deviation of ± 1.4% and had a detection limit of approximately 50 femtomoles. Improvements in the stability and fluorescence response of lysine and hydroxylysine were obtained by the incorporation of sodium dodecyl sulfate in the derivatizing medium. Applications of the chromatography system involving the amino acid analysis of peptides after either acid or enzymatic hydrolysis are presented. Methods for the sequence analysis of picomole quantities of peptides by time course hydrolysis with exopeptidases were also developed, which employed the above separation procedure for the identification and quantifica...

David F. Johnson

Papers

The structure of the fluorescent adduct formed in the reaction of o-phthalaldehyde and thiols with amines

Reaction of o-phthalaldehyde and thiols with primary amines: formation of 1-alkyl(and aryl)thio-2-alkylisoindoles

Reaction of o-phthalaldehyde and thiols with primary amines: fluorescence properties of 1-alkyl(and aryl)thio-2-alkylisoindoles.

Ethanethiol: a thiol conveying improved properties to the fluorescent product of o-phthalaldehyde and thiols with amines.

The structure of the fluorescent adduct formed in the reaction of o‐phthalaldehyde and thiols with amines