Advances in the mass spectrometry of hemoglobin adducts: global analysis of the covalent binding of butadiene monoxide.

A common method to assess exposure to 1,3-butadiene through both occupational and environmental routes involves the detection of hemoglobin adducts formed by the primary reactive metabolite butadiene monoxide (EB). This assay is a modification of the Edman degradation procedure, which was developed to determine adducts formed specifically at the amine group of the N-terminal valine of hemoglobin. The goals of the current research are to determine the global modification of alpha- and beta-globin chains by EB and to localize the primary reactive residues to specific regions of the globin polypeptides. The degree of modification was monitored by electrospray mass spectrometry, which was used to measure the formation of EB-hemoglobin adducts (up to ten adducts per globin). Structural analysis of these modifications was performed by peptide mapping of globin peptides after trypsin digestion using liquid chromatography-mass spectrometry. These experiments provided information as to the relative reactivity of alpha- and beta-globin towards EB, as well as to the localization of adducts to specific peptide sequences. The results reveal variable reactivities of alpha- and beta-globin towards EB and also show the formation of multiple adducts at several alpha- and beta-globin sites. In addition, it is established that the N-terminal valine residues are not the first to be modified by EB.

A comprehensive structural analysis of hemoglobin adducts formed after in vitro exposure of erythrocytes to butadiene monoxide.

A widely used method for assessing occupational and environmental exposure to 1,3-butadiene involves the detection of hemoglobin adducts formed by the reactive metabolite butadiene monoxide (BMO). This assay employs the N-alkyl Edman method, which was developed to determine adducts formed at the amine group of the N-terminal valine of hemoglobin. Disadvantages of this procedure include its limitation to detecting only one adduct per globin chain, despite the presence of numerous other, and potentially more reactive, nucleophilic amino acids in hemoglobin. The method is also not suitable for determining whether the reaction of BMO occurs at the N-terminal valine of alpha- or beta-globin. The primary goals of the current research are to determine the degree of modification of alpha- and beta-globin chains by BMO and to localize the reactive residues to specific regions of the globin polypeptides. The reaction products after in vitro incubation of C57Bl/6 mouse erythrocytes with BMO were isolated by acid extraction of heme and microprecipitation of globin, followed by the determination of the number and location of adducts by mass spectrometry. The modification degree was monitored by electrospray mass spectrometry, which was used to measure the time- and concentration-dependent formation of BMO-hemoglobin adducts (< or =10 adducts per globin). The results indicate that BMO reacts faster and to a higher degree with alpha-globin than with beta-globin. Structural analysis was performed by peptide mapping of globin peptides after trypsin digestion using liquid chromatography/mass spectrometry. These experiments allowed the localization of BMO-hemoglobin adducts to specific regions within alpha- and beta-globin, and also provided information about their relative reactivity. Interestingly, the initial site of adduct formation on alpha-globin is located near the N-terminal peptide, whereas the initial site on beta-globin is located at the C-terminal region. Collectively, the results establish differences in the reactivities of alpha- and beta-globin toward BMO, demonstrate the formation of multiple adducts at several alpha- and beta-globin sites, and show that the N-terminal valine residues are not the first to be modified by BMO.

Characterization of the reactivity, regioselectivity, and stereoselectivity of the reactions of butadiene monoxide with valinamide and the N-terminal valine of mouse and rat hemoglobin.

Occupational exposure to 1,3-butadiene (BD) has been monitored by measuring the level of hemoglobin N-terminal valine adduct formation with the primary reactive metabolite, butadiene monoxide (BMO). However, mechanistic details concerning the relative reactivity, regioselectivity, and stereospecificity of BMO with the N-terminal valine of hemoglobin are lacking. In the studies presented here, L-valinamide was used as a model for the N-terminal valine of hemoglobin to compare the nucleophilic reactivity, regioselectivity, and stereoselectivity of the reaction both in aqueous solution and within a protein microenvironment. Four products produced by the reaction of L-valinamide with racemic BMO (two pairs of diastereomers produced by reactions at C-1 and C-2 of the epoxide moiety) were synthesized, purified, and characterized by (1)H NMR and GC/MS. These four reaction products were used as analytical standards for kinetic studies of the reaction of valinamide with BMO at physiological pH (7.4) and temperature (37 degrees C). The results show that the adducts formed by reaction at C-2 were formed at a ratio of approximately 2:1 compared to the adducts formed by reaction at C-1. The stereoisomers of each respective regioisomer were produced with similar rates of formation. The reaction of BMO with the N-terminal valine of hemoglobin was also studied in vitro using intact erythrocytes from Sprague-Dawley rats and B6C3F1 mice. After cleavage of the N-modified valine by the N-alkyl Edman degradation procedure using pentafluorophenylisothiocyanate (PFPITC), a novel procedure was developed that allowed GC/MS detection and quantitation of the four expected products by silylation of the PFPTH-valine-BMO derivatives. The hemoglobin results contrast with the valinamide results in that the reaction of BMO with the N-terminal valine residue in both rat and mouse hemoglobin produced mostly C-1 adducts. The rates obtained with rat hemoglobin were much slower than the rates obtained with mouse hemoglobin or with valinamide. These results, and the finding that the reaction with rat hemoglobin produced a higher ratio of C1:C2 adducts in comparison with the reaction with mouse hemoglobin, indicate the importance of measuring all four adducts when comparing the relative rates of adduct formation both with model compounds and among different species.

Method for measuring the activities of cholesteryl ester transfer protein (lipid transfer protein).

A continuous recording fluorescence assay was developed for cholesteryl ester transfer protein (CETP). The assay measures the increase in fluorescence accompanying the relocation of fluorescent lipids, cholesteryl esters and triglycerides, from a donor emulsion to an acceptor emulsion. In the absence of CETP, the quantum yields of the fluorescent lipids is low because their high concentrations in the donor emulsions result in self-quenching. CETP catalyzes the redistribution of the fluorescent lipids from the donor to the acceptor emulsions and fluorescence increases substantially. Efficient sonication and incorporation of apolipoproteins from human HDL into the emulsions significantly increased the transfer rates. Under optimal conditions, the redistribution of fluorescent compounds reaches equilibrium within < 30 min and the kinetics of this process are consistent with a simple, first-order reaction pathway. The redistribution kinetics support a mechanism of adsorption --> exchange --> desorption --> diffusion.

Semisynthetic proteins: model systems for the study of the insertion of hydrophobic peptides into preformed lipid bilayers.

Models of protein translocation and secretion will not be complete without details of the mechanism of lipid bilayer insertion. The study of spontaneous hydrophobic peptide interactions with model membrane systems has been hindered by their very low solubility in aqueous solutions. A novel protocol has been developed that enables the site-specific (N-terminus) attachment of hydrophobic peptides to a water-soluble carrier protein [bovine pancreatic trypsin inhibitor (BPTI)] using a heterobifunctional crosslinker (SPDP). In this initial study H-(Ala)20-Tyr-Cys-CONH2 and H-(Ala)10-Tyr-Cys-CONH2 were selected as hydrophobic peptides, since alanine is the simplest alpha-helix-forming amino acid, and the peptides as alpha-helices are just long enough to span the lipid bilayer and monolayer, respectively. The carrier protein was treated with sigma-methylisourea, which resulted in the guanidination of the four lysine epsilon-amine groups. The chemical modification of BPTI to give G-BPTI allowed the attachment of SPDP specifically to the free N-terminal alpha-amine group. The peptides were synthesized with a C-terminal cysteine moiety, allowing the site-specific cross-linking of the peptides to the N-terminus. In order to prevent peptide aggregation, the synthetic peptides were cleaved from the preparative resin in detergent and cross-linked to G-BPTI. After cross-linking, the detergent was removed from the mixture by gel filtration employing propionic and formic acids in the mobile phase. The detergent-free, peptide--G-BPTI conjugates were subsequently purified by reversed-phase HPLC. The interaction parameters of the two semisynthetic proteins with large unilamellar vesicles were determined by ultracentrifugation of the equilibrated vesicle--protein mixtures. For comparison, the same semisynthetic proteins were reconstituted into lipid vesicles using an octyl glucoside dilution technique. The incorporation and reconstitution data proved to be quite similar. The results indicated that (Ala)20--G-BPTI interacted with LUV to form a stable complex and behaved as a membrane protein in reconstituted bilayer systems. (Ala)10--G-BPTI, however, remained in the aqueous phase in both bilayer systems. The thermodynamic interaction data are compared to the theoretical values of total free energy changes calculated for the incorporation of model hydrophobic alpha-helices. In addition, the solubility and stability of the hydrophobic peptides, both in the aqueous phase and membrane-bound, were studied by cleaving the disulfide bond linking the peptides to G-BPTI using dithiothreitol. Molecular sieve chromatography was used to evaluate the state of self-association of the semisynthetic proteins in aqueous solutions.