Analytical Biochemistry

A method derived from DNA melting-curve analysis has been modified for determining homogeneity of DNA species centrifuged to equilibrium in a CsCl gradient. DNA species of known density were purified, centrifuged to equilibrium in a Spinco Model E analytical ultracentrifuge, and analyzed by probability distribution. In order to determine the limits of resolution by this procedure, samples with varying buoyant-density separations were obtained by mixing different DNA species. Results indicate this method can detect overlapping mixtures of DNA in relative amounts of 9:1 and resolve two species of DNA of equal concentrations with a buoyant-density separation of 0.001 g/cm3.

A simple method of high-pressure liquid chromatography for the determination of cortisol in 0.1 ml of human serum is described. Multiple steps of the previous procedures can be simplified by shaking a glass rod for effective extraction of hormones and by performing all procedures on a microscale. The recovery rate was better than that of the previous method and the coefficient of variation was 5.3%. The present method which has a limit of detection of 1–30 ng is particularly useful for the assay of serum cortisol in infantile patients.

The sensitivity of ATP determinations with crude firefly luciferin luciferase is limited by contaminating ATP converting enzymes, which cause a rapid decrease of the ATP level during the assay. Purified luciferase has the advantage of producing an almost constant light intensity proportional to the ATP concentration. Sensitivity and specificity of the ATP assay are, therefore, considerably increased when purified enzyme is used instead of crude extracts of the enzyme. ATP, 0.1–1.0 pmol as well as higher amounts can be determined with commercial preparations of purified and stabilized luciferase. In ADP and AMP measurements with the luciferase assay, problems are arising from the enzymes required for the conversion to ATP, since they are frequently contaminated by low amounts of adenine ribonucleotides. Exclusion of contaminated enzymes and removal of ammonium sulfate from adenylate kinase were the only prerequisites for determinations of 0.1–1.0 pmol of ADP and AMP with purified luciferase. The application of the assay in determinations of ATP, ADP, and AMP in single preimplantation mouse embryos is described.

A review of the modern literature reveals that the values for quinine fluorescence lifetime are in good agreement, the mean value being 18.91±0.56 nsec. By virtue of some very unusual properties, quinine appears suitable for use as a lifetime reference standard for any value from 0.189 to 18.9 nsec, with an expected accuracy of ±3% throughout this range. Cl−, not normally considered a quenching agent, quenches quinine emission at diffusion-controlled rates. The Stern-Volmer plot was unique in that the strict lincarity, indicating pure collisional quenching, was maintained even when fluorescence was > 99% quenched. Thus, solutions of quinine-NaCl can be made up having lifetimes known to great accuracy. Similarly, γ-pyrenebutyrate solutions containing KI are suitable standards for the range up to 115 nsec. The compositions of such solutions have been calculated and tabulated. It is argued that the lifetimes of these solutions are at least as reliable as any of the hundreds of lifetimes which have been reported in the literature. Several important applications of such lifetime standards are discussed.

Reactive oxygen species (ROS) play important roles in the defense mechanism against infection and in the pathogenesis of various diseases. Although chemical properties of ROS generated by leukocytes have been studied extensively, methods available for their analysis are not sufficiently sensitive. We found that 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione (L-012) reacted with various types of ROS generated by activated neutrophils in human blood and oral cavity, and from peritoneal cavity of the rat, and developed strong chemiluminescence (CHL). Under physiological conditions, opsonized zymosan-dependent CHL intensity of L-012 in human blood and rat peritoneal neutrophils was about 100 and 10 times higher than that of luminol and luciferin analog MCLA, respectively. Phorbol ester-activated CHL of oral neutrophils was also higher with L-012 than that with luminol and MCLA. The presence of either superoxide dismutase, catalase, uric acid, deferoxamine, or azide decreased CHL intensity of L-012 by 52, 57, 57, 63, and 91%, respectively. Kinetic analysis revealed that L-012 developed CHL predominantly by reacting with hydroxyl radical and hypochlorite. Thus, highly sensitive L-012 permits studies on ROS generation by complex biological systems, such as leukocytes, and on the role of ROS in the pathogenesis of various diseases.

During the investigations of the metabolic pathways of the new dopaminergic drug N-0437 we encountered a substantial difference in HPLC-retention times between the metabolites, detected by a uv spectrophotometer, and their tritium-labeled markers, measured off-line by a scintillation counter. These distinct retention times can be ascribed to a phenomenon known as isotopic fractionation. In this article we quantified the isotopic separation by reversed-phase HPLC of the unlabeled N-0437, its deuterated and tritiated analogs, and their corresponding glucuronides, synthesized in vitro by rat liver microsomes. In the separation of the glucuronides we demonstrated that this isotope effect is dependent largely on the eluent pH.

Methods for the activation of a cellulose dialysis membrane for immunosensor applications have been developed. For activation two reagents, 1,1'-carbonyldiimidazole (CDI) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), were compared with respect to the coupling efficiency for glucose oxidase (GOx) and 1,8-diamino-2,6-dioxaoctane. The maximum level of activation was 2.4 micromol cm(-2) for CDI and 0.2 micromol cm(-2) for CDAP activation. We observed 1.5 microg cm(-2) and 0.4 x 10(-4) U cm(-2) GOx with CDI-activated membranes whereas 1.7 microg cm(-2) and 7.2 x 10(-4) U cm(-2) GOx were observed with CDAP-activated membranes. With 1,8-diamino-2,6-dioxaoctane amino group densities of 0.165 and 0.09 micromol cm(-2) were observed via CDI and CDAP activation, respectively. An amino-modified membrane was used for coupling a ligand (pentapeptide) and an immunoenzymometric assay for hemoglobin A1c was carried out.

Water-soluble 2-hydroxypropyl-beta-cyclodextrin (hp-beta-CyD), a cyclic and nonreducing oligosaccharide, was used to enclose the hydrophobic guest molecules ferrocene (FeCp2) and 1,1'-dimethylferrocene (DMFeCp2) to form a water-soluble complex. At high concentrations (300 mM), hp-beta-CyD enclosed up to 100 mM FeCp2 or DMFeCp2. The yellow complexes were electrochemically oxidized (platinum vs Ag/AgCl poised at +450 mV) to form the blue dyes ferricinium (FeCp2+) and 1,1'-dimethylferricinium (DMFeCp2+). This is a one-electron transfer process and the ferricinium cations formed exhibited absorption peaks at 620 and 650 nm, respectively. The concentrated DMFeCp2+ was much more stable (4 months) than the FeCp2+ (2 days) and both oxidized dyes were insensitive to a wide pH variation (pH 2-11). The DMFeCp2+ was reduced easily by various reducing agents such as ascorbic acid, uric acid, and sulfite and exhibited an absorption coefficient of 325 cm-1 M-1. Application of the novel DMFeCp2+ complex as a colorimetric dye for the enzymatic oxidation of glucose, glutamate, lactate, phenylalanine, xanthine, and hypoxanthine was successfully demonstrated. When applied to real samples the results obtained agreed well with those of standard enzymatic assays. The DMFeCp2+ complex could also be utilized to monitor activity of oxidases under saturating substrate concentrations.

Metabolism of S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC) yields chlorofluorothioacetyl fluoride, which reacts with cellular proteins to form stable lysine adducts. Little is known about the subcellular localization of these protein adducts or about their role in CTFC-induced nephrotoxicity. A method for the synthesis of CTFC and other cysteine S-conjugates labeled with 3H at the S-alkyl or S-alkenyl position would be useful in studies of S-conjugate metabolism and toxicity. Reaction of L-cysteine, chlorotrifluoroethene, 1,8-diazabicyclo[5.4.0]undec-7-ene, and 3H-labeled water followed by repeated crystallization yielded radiochemically pure [3H]CTFC (235 mg, 20% yield; sp act 1.07 x 10(9) Bq/mmol), which was identical to CTFC by TLC, 1H NMR, and 19F NMR. 3H NMR revealed a doublet of triplets at 6.5 ppm with geminal and vicinal T-F couplings of 51.5 and 6.0 Hz, respectively, consistent with the proposed structure. When 2H-labeled water was used, [2H]CTFC was formed, and its structure was confirmed by 1H and 19F NMR, FAB-MS, and TLC. Analysis of renal and hepatic subcellular fractions of rats given 1, 10, or 100 mumol/kg [3H]CTFC showed a dose-dependent binding of 3H-containing metabolites to liver and kidney proteins.

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) has been adapted for use as a coupling reagent for tert-butyloxycarbonyl (Boc) amino acids in automated solid-phase peptide synthesis. When compared to the existing preformed symmetrical anhydride procedure employing dicyclohexyl-carbodiimide (DCC), the use of TBTU in the presence of 1-hydroxybenzotriazole (HOBt) provides a more efficient coupling procedure for Boc-amino acid derivatives. Overall cycle times using TBTU/HOBt coupling reagents (30 min) compare favorably to those of the DCC-mediated procedure (approx 65 min). Dimethylformamide can be used as the sole solvent for both activation and coupling reactions. Implementation of TBTU/HOBt coupling conditions does not require replumbing of any lines of the Applied Biosystems Model 430A instrument and necessitates changes to only three reagent bottle positions. The variable coupling efficiencies of Boc-asparagine following activation with TBTU/HOBt (as low as 89%) can be overcome by protection of the amide function of Boc-asparagine with the 9-xanthyl group. Examples of the synthesis and characterization of a number of peptides ranging in length from 13 to 29 residues are given.

Constant wavelength synchronous fluorescence spectroscopy (CW-SFS), UV-visible absorption spectroscopy, and cyclic and differential pulse voltammetry were applied to investigate the competitive interaction of DNA with the bis(1,10-phenanthroline)copper(II) complex cation ([Cu(phen)(2)](2+)) and a fluorescence probe, neutral red dye (NR), in a tris-hydrogen chloride buffer (pH 7.4). The results show that both the [Cu(phen)(2)](2+)and the NR molecules can intercalate competitively into the DNA double-helix structure. The cyclic voltammetry method showed that both anodic and cathodic currents of [Cu(phen)(2)](2+) decreased on addition of the DNA and the intercalated [Cu(phen)(2)](2+)-DNA complex formed (beta = (4.14 +/- 0.24) x 10(3)). CW-SFS measurements were facilitated by the use of the three-way resolution of the CW-SFS for NR, [Cu(phen)(2)](2+), and NR-DNA. The important constant wavelength (CW) interval, Deltalambda, was shown to vary considerably when optimized (135, 58, and 98 nm for NR, NR-DNA, and [Cu(phen)(2)](2+), respectively). This approach clearly avoided the errors that otherwise would have arisen from the common assumption that Deltalambda is constant. Furthermore, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way CW-SFS data, and the results provided simultaneously the concentration information for the three reaction components, NR, [Cu(phen)(2)](2+), and NR-DNA, for the system at each equilibrium point. The PARAFAC analysis indicated that the intercalation of the [Cu(phen)(2)](2+) molecule into the DNA proceeds by exchanging with the NR probe and can be attributed to two parallel reactions. Comprehensive information was readily obtained; the replacement of the intercalated NR commenced immediately on introduction of [Cu(phen)(2)](2+), approximately 50% of NR was replaced by [Cu(phen)(2)](2+) at a concentration of 0.45 x 10(-5) mol L(-1), and nearly all of the NR was replaced at a [Cu(phen)(2)](2+) concentration of 2.50 x 10(-5) mol L(-1). This work has the potential to improve extraction of information from the fluorescence intercalator displacement (FID) assay.

An equation is found relating the fractional activity, (v/v0), of an enzyme assay mixture to the total concentrations of metalloenzyme, active site metal ion, metal-binding ligand and substrate and the stability constants of the complexes present. When (v/v0) is measured as a function of the total ligand concentration, this equation offers a way of data-plotting which yields straight lines and permits the calculation of the metal-binding constant KME from either the slope or the intercept, provided that mixed complexes (enzyme-metal ion-ligand) do not contribute significantly to the change in (v/v0). Since deviations from linearity occur in the latter case, the proposed inhibition plot serves as a diagnostic tool for the recognition of such complexes. Application to the inhibition of thermolysin by 1,10-phenanthroline gives a value of 2.1 × 1011m−1 for KZnE, the binding constant of the active site zinc ion, at pH 7.50, 25°C and ionic strength 0.1. The equation also allows the rapid calculation of the ligand concentration necessary to attain a desired degree of inhibition when the total enzyme and active site metal ion concentrations of the solution are known.

A method was developed for the synthesis of stereo-specific sn-1,2-diacylglycerols radioactively labeled on the glycerol backbone. This method includes the acylation of L-[U-14C]glycerol 3-phosphate with fatty acid anhydrides in the presence of dimethylaminopyridine and the tetraethylammonium salt of the fatty acid used for the acylation. The phosphatidic acid obtained from this synthesis was converted to sn-1,2-diacyl[U-14C]glycerol by the action of phospholipase C. When di10:0-, di-18:1-, di-18:2-, and di-22:1-diacyl[U-14C]glycerols made by this method were provided as substrates to microsome preparations from developing cotyledons of safflower (Carthamus tinctorius), they were all incorporated into phosphatidylcholine by the action of cholinephosphotransferase. The time course of the incorporation into phosphatidylcholine showed no differences for these four substrates and the initial rates were comparable.

Operational advantages in using CPA for the determination of enzymic azo reduction are described. Enzyme activity of the 9000g supernatant fractions of liver, with CPA as the substrate, demonstrated characteristics similar to those previously reported with other azo compounds as substrates.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol; CDP-DG) is an important intermediate in the biosynthesis of the major glycerophosphate-based phospholipids of prokaryotes and eukaryotes. This compound is expensive to purchase and inefficient to prepare chemically. Radiolabeled CDP-diacylglycerol is unavailable commercially. We describe a simple and inexpensive method to synthesize [3H]CDP-DG enzymatically. The three-step enzymatic procedure includes phosphorylation of [3H]glycerol to sn-[3H]glycerol 3-phosphate (G3P) by glycerokinase,acylation of [3H]G3P to [3H]phosphatidic acid (PA) by G3P acyltransferase, and conversion of [3H]PA and CTP to [3H]CDP-DG by CDP-DG synthase. This procedure is considerably less labor intensive and less expensive than is chemical synthesis, and the yield is at least 30%.

High-performance liquid (HPLC) and thin-layer (TLC) chromatographic methods for the detection and quantification of diazeniumdiolates are described. The HPLC determinations were made using a Rocket Platinum NH2 column (7 x 53 mm, 100-A pore size, 3-microm particle size), under isocratic conditions with mixtures of acetonitrile, methanol, and water containing 0.1% diethylamine (v/v), at a flow rate of 2.5 ml/min, a column temperature of 22 degrees C, and UV detection at 250 nm. The TLC determinations were similarly made using Merck NH2 F254S precoated glass plates (approximately 2 x 5 cm, 5-microm particle size, 0.2-mm layer thickness) with mixtures of acetonitrile, methanol, and water containing 0.1% diethylamine (v/v). Preexisting traces of carcinogenic nitrosamines were detected in some samples of diazeniumdiolates. The methods provide a more efficient means of characterizing the purity of diazeniumdiolate samples prepared for use in biomedical applications than older procedures which rely on differential absorbance measurements at 250 and 350 nm, respectively.

An automated alkaline elution system for the detection of DNA damage has been developed. After manual application of samples, which is completed within 5 min, the subsequent supply of liquids, changes in flow rates, and temperature are controlled automatically. The system operates 16 filters and may easily be expanded. The sensitivity of the fluorometric DNA determinations with the Hoechst 33258 dye is increased by using an elution buffer (20 mM Na2EDTA, pH 12.50) with low background fluorescence. DNA is determined using an automated setup similar to the one recently presented by Sterzel et al. (1985, Anal. Biochem. 147, 462-467). The most significant modification is the use of a neutralization buffer which allows variations in the pH of eluted fractions. This change increases the sensitivity of the DNA measurements. The automated alkaline elution system was evaluated using the nematocide 1,2-dibromo-3-chloropropane (DBCP) in a study of its genotoxic effects in the testes and the kidneys. Significant DNA damage was induced in testicular cells by 2.5 microM DBCP (1 h) in vitro and 85 mumol/kg DBCP ip (3 h) in vivo. The damage appeared after short treatment times (10 min in vivo). Variations in the observed DBCP response in vivo were largely due to interanimal variations. The automated alkaline elution system proved to be a sensitive assay also for the detection of DNA damage in kidney nuclei prepared from rats exposed to DBCP. Provided that kidney nuclei from untreated rats, mice, or hamster were kept ice-cold until lysing, 85-100% of their DNA was retained after 16 h of elution, indicating highly intact DNA. Under the same conditions, guinea pig DNA was rapidly degraded unless the nuclei were prepared in a buffer with a higher concentration of Na2EDTA (20 mM).

An extremely sensitive fluorimetric HPLC method used to determine in vivo cerebral alpha-ketoisocaproic acid (KIC) concentration with less than 2 g of tissue (e.g., a single rat brain, approximately 1.8 g) is reported. Removal of unwanted lipids and amino acids and isolation of alpha-ketoacids in an optimal derivatization buffer are achieved by C18 solid-phase extraction of the acid-soluble fraction of brain tissue. Quantitation of KIC to the femtomole level is accomplished by reversed-phase HPLC using 4,5-dimethoxy-1,2-diaminobenzene precolumn fluorescence derivatization and on-line fluorescence detection. These techniques are applicable to femtomole quantitation of other alpha-ketoacids in various tissue and blood matrices. Combination of this fluorimetric method with simple nonchromatographic procedures to measure in vivo cerebral [1-14C]alpha-ketoisocaproic acid radioactivities in tissue provides estimates of specific activities.

A recently developed UV spectroscopic method for quantitating isothiocyanates (R-N=C=S) at the nanomole level is based on the observation that the highly electrophilic central carbon atom of the -N=C=S group can undergo successive nucleophilic additions with reagents containing two sulfhydryl groups on adjacent carbon atoms to form a cyclic thiocarbonyl product and release the nitrogen atom as a primary amine (Y. Zhang, C.-G. Cho, G. H. Posner, and P. Talalay, Anal. Biochem. 205, 100-107, 1992). The assay utilizes 1, 2-benzenedithiol as the vicinal dithiol reagent and measures the reaction product, 1,3-benzodithiole-2-thione, spectroscopically (am of 23,000 M-1 cm-1 at lambdamax = 365 nm). This paper reports a dramatic improvement in the analytical sensitivity of this method. By separating the cyclocondensation product by a simple isocratic HPLC method and using an automatic integrator, the sensitivity of detection has been lowered to a few picomoles of isothiocyanate. Furthermore, we now find that the chemical specificity of the cyclocondensation reaction is not restricted to isothiocyanates, but includes dithiocarbamates, and related thiocarbonyl compounds such as carbon disulfide, certain substituted thiourea derivatives, and xanthates. The availability of such analytical methods is important not only because isothiocyanates (and their glucosinolate precursors) are present in edible plants and are consumed by humans in substantial quantities, but also because some dithiocarbamates are toxic, and are widely used in the rubber industry as vulcanization accelerators and in agriculture as fungicides, insecticides, and herbicides. The analysis of many isothiocyanates is complicated by their extreme volatility. This difficulty can be circumvented by converting isothiocyanates quantitatively into dithiocarbamates (by facile addition of a mercaptan such as N-acetylcysteine) and quantitating the nonvolatile dithiocarbamate by the cyclocondensation reaction.

Processes for enhancing the chemiluminescent signal generated by enzyme-activated 1,2-dioxetanes have been developed for membrane-based assays in which detection is done using a charge-coupled device (CCD) camera or X-ray film. The enhancement is demonstrated using slot-blots of biotinylated lambda DNA in conjunction with an avidin-alkaline phosphatase conjugate. For detection with a CCD camera, the nylon membrane is dried after processing and incubation in dioxetane substrate solution and heated to temperatures of 50 to 80 degrees C during detection. Up to a 100-fold signal increase is obtained using this enhancement process compared to the conventional detection procedure, in which the blot is kept saturated with substrate solution in a sealed plastic bag during detection. For detection with X-ray film, a fivefold increase in signal intensity is realized by drying the membrane before exposure to the film. These enhancement processes greatly reduce the time required for detection in membrane-based assays.

In providing chemiluminescent probes that have high chemiluminescence intensity and high specificity to superoxide anions, novel chemiluminescent probes involving cyclodextrins covalently bound to 6-(4-methoxyphenyl)imidazo[1,2-alpha]pyrazin-3(7H)-one with fluorescein were synthesized and characterized. Using the hypoxanthine-xanthine oxidase system for the generation of the superoxide anions, these novel chemiluminescent probes showed higher superoxide-induced chemiluminescence intensity than that of 6-[4-[2-[N(')-(5-fluoresceinyl)thioureido]-ethoxy]phenyl]-2-methylimidazo[1,2-alpha]pyrazin-3(7H)-one (FCLA). When tested at a probe concentration of 1.0 microM, compound 6, in which 6-(4-methoxyphenyl)imidazo[1,2-alpha]pyrazin-3(7H)-one and fluorescein are covalently attached on the secondary and primary hydroxyl faces of gamma-cyclodextrin, respectively, showed green luminescence intensity that was 26 times that of FCLA, which was also the highest luminescence intensity in this present study. At probe concentrations of less than 1.0 microM, the ratio of the superoxide-dependent chemiluminescence intensity to the background chemiluminescence intensity for compound 6 was higher than that of FCLA. This high superoxide-induced chemiluminescence intensity and superoxide specificity in low probe concentrations indicates that 6 can be more effective than FCLA toward the measurement of superoxide anions.

An HPLC-fluorescence assay has been developed for the determination of the activity of rat renal cytosolic cysteine conjugate beta-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid, specific, and very sensitive. The assay has been evaluated with two model substrates for rat renal cytosolic beta-lyase, notably S-1,2-dichorovinyl-L-cysteine (DCVC) and S-2-benzothiazolyl-L-cysteine (BTC). Equimolar formation of pyruvic acid and 2-mercaptobenzothiazole, a chromophoric thiol, indicated that pyruvic acid formation actually reflects the beta-elimination activity of beta-lyase during the beta-elimination of BTC. From this it follows that the pyruvic acid assay can be applied to the measurement of the beta-elimination activity of this enzyme, independent of the presence of chromophoric groups or radiolabels in substrates. Due to the large linear range and the very high sensitivity of the present HPLC-fluorescence assay (detection limit, 7.5 pmol of pyruvic acid), both good and poor substrates of beta-lyase can be measured. Enzyme kinetic data are presented for the model substrates BTC and DCVC and for four structurally related S-2,2-difluoroethyl-L-cysteine conjugates.

An extremely sensitive and specific analytic procedure is described for quantitating 1,2-diacylglycerol (DAG). Escherichia coli DAG kinase (EC 2.7.1.-) catalyzed the formation of [32P]phosphatidic acid by the transfer of 32PO4 from [γ-32P]adenosine 5′-triphosphate to DAG in linear proportion to the quantity of added DAG from 10 to 1000 pmol. This technique allowed reliable detection of as little as 2 pmol of added DAG. To assess levels of DAG in tissue lipid extracts a miniaturized method for silicic acid column chromatography was developed to separate DAG from triglycerides and phospholipids. When these procedures were applied to erythrocytes, lysis in the presence of Ca2+ caused a 10.6-fold rise in cellular DAG confirming not only the results obtained in an earlier investigation (1), but also the utility of this technique in the analysis of exceedingly small quantities of cellular DAG.

The probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) is widely used for studying the superoxide anion production and the efficiency of antioxidants in biological systems. Here we report that a number of sulfur-containing compounds applied in biochemical and cytological studies are able to suppress MCLA-derived chemiluminescence (MDCL) independent of their capability to scavenge superoxide anion. The most effective MDCL quenchers appeared to be the substances with thiocarbamoyl and thiocarbonyl groups coupled to cyclic molecules and several thiol- and disulfide-containing compounds. The analysis of MDCL kinetics in a xanthine oxidase system allows one to rapidly discriminate between true antioxidants and the quenchers of chemiluminescence.

N-(Fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (F-DHPE) is a lipid fluorescence dye sensitive to pH changes and is used in this study for detecting proton flux through F0F1-ATPase within chromatophores driven by ATP hydrolysis. F-DHPE is easily labeled to the outer surface of chromatophores. In the range of pH 7.0 to 9.0, fluorescence intensity is sensitive to pH changes. The sensitivity is especially great in the range of pH 8.2 to 9.0, so pH 8.6 was chosen as the appropriate experimental condition. It is shown that added ATP not only acts as a fluorescence quencher but also can be hydrolyzed by F0F1-ATPase to pump protons into chromatophores, resulting in fluorescence restoration. A stimulator (NaSO3) and various types of inhibitors (NaN3, 5'-adenylyl imidodiphosphate [AMP-PNP], and N,N'-dicyclohexylcarbodiimide [DCCD]) of F0F1 confirmed that fluorescence restoration is caused by ATP-driven proton flux. When loaded with one antibody (anti-beta antibody) or two antibodies (anti-beta antibody and sheep to rabbit second antibody), F0F1-ATPase exhibits lower proton pumping activities, as indicated by fluorescence restoration. The possible mechanism of the inhibition of antibodies on proton pumping activity is discussed.

A highly sensitive silver stain for visualizing proteins in polyacrylamide gels was adapted for staining 1,2-diol groups of carbohydrates. This periodic acid-silver stain is 64 times more sensitive than the periodic acid-Schiff stain, and the lower limit of detection is about 0.4 ng of bound carbohydrates. Its lower limit of detection is comparable to autoradiography of radioactively labeled lipopolysaccharides.

To determine 13C enrichment at specific positions of glucose, plasma samples (50-100 microliters) were deproteinized using ethyl alcohol, centrifuged, and evaporated to dryness, and the residues were derivatized with acetone/H2SO4 (100/1, v/v). After acetylation, the 1,2:5,6-diisopropylidene-3-O-acetyl-alpha-furanosyl derivative was analyzed by gas chromatography-mass spectrometry. The method was tested using mixtures of natural and 13C-labeled glucose molecules. Ions at m/z 113, 114, 143, 144, 287, and 288, corresponding to the C2-C4, C1-C4, and C1-C6 parts of natural or 13C-labeled glucose molecule, respectively, were selectively monitored to determine the 13C content of these fragments. This method was applied to the study of plasma glucose-labeling pattern following the infusion of [3-13C]lactic acid at tracer doses. Assuming equilibration of label through triose-phosphate isomerization in vivo, we determined the 13C enrichment of carbons 1, 2, and 3 of glucose; the values for E1/E3 and E2/E1 ratios of 8.1 and 0.77, respectively, are consistent with those obtained in studies using radioactive tracers.

Appreciable amounts of inositol 1,2-cyclic 4,5-trisphosphate (cIP3) are formed on agonist stimulation of secretory cells, e.g., pancreas (1,2) and parotid (3,4). However, the physiological role of this compound is unknown. To obtain sufficient amounts of cIP3, we have developed a synthetic method to produce cIP3 from inositol 1,4,5-trisphosphate (I(1,4,5)P3). The method is an adaptation of the dicyclohexylcarbodiimide (DCCD) method of Khorana et al. (5), which was originally developed to synthesize 2',3'-cyclic ribonucleotides. The method involves treatment of the pyridinium salt of I(1,4,5)P3 with DCCD in pyridine water, which cyclizes part of the 1-phosphate on the inositol ring to the 1,2-cyclic phosphate. The compound identified as cIP3 cochromatographed with authentic cIP3 in two HPLC systems and on ionophoresis. It was converted to I(1,4,5)P3 on mild acid treatment--a characteristic of cyclic inositol phosphates. Inositol 1,2-cyclic 4,5-trisphosphate is then purified by HPLC. Sufficient amounts of cIP3 can be prepared by this method to carry out numerous experiments on its possible cellular role.

We developed gas chromatographic-mass spectrometric assays for the enantiomers of 1,2-propanediol, 1,3-butanediol, 1,3-pentanediol, and their corresponding hydroxyacids, lactate, beta-hydroxybutyrate, and beta-hydroxypentanoate (3-hydroxyvalerate) in biological fluids. The corresponding ketoacids, acetoacetate and beta-ketopentanoate, can be assayed simultaneously by pretreating the samples with NaB2H4. The assays involve spiking the samples with deuterated internal standards, deproteinization, ether extraction, and derivatization of the carboxyl groups with (R,S)-2-butanol/HCl and of the hydroxyl groups with chiral (S)-(+)-2-phenylbutyryl chloride. Mass spectrometric analysis is conducted under ammonia positive chemical ionization. We used these assays to follow the metabolism of diol enantiomers in dogs. For (R,S)-1,3-butanediol and (R,S)-1,3-pentanediol, the uptakes from dog plasma of the R and S enantiomer of each diol were identical. In contrast, the metabolism of (S)-1,2-propanediol was faster than that of (R)-1,2-propanediol. (R)-1,2-Propanediol is formed during acetone metabolism, while (R,S)-1,3-butanediol and (R,S)-1,3-pentanediol are potential nutrients. The assays developed will allow further investigations of the metabolisms of acetone, (R)-lactate, and artificial nutrients derived from the 1,3-butanediol and 1,3-pentanediol enantiomers.

A simple and rapid method for the quantitation of proteins separated either by sodium dodecyl sulfate-electrophoresis or by isoelectric focusing in slab gels is presented. The method is based on the solubility of polyacrylamide gels crosslinked with N, N'-1, 2-dihydroxyethylenebisacrylamide (DHEBA) in periodic acid. After electrophoretic separation proteins are stained with Coomassie brilliant blue G-250. DHEBA gels show considerable swelling during the staining and destaining process but can be shrunk to their normal size in a 10% (w/v) solution of ammonium sulfate. Stained bands are cut from the gel and solubilized in periodic acid. During dissolution the dye decolorizes. Protein concentration in the solution is determined by a modified Coomassie dye-binding assay. Quantitation is linear in the range of 100 ng to 5 micrograms and not disturbed by dissolved gel. Separations in N, N'-1, 2-dihydroxyethylenebisacrylamide-crosslinked gels show qualities similar to those in normal crosslinked gels.

A procedure for the assay of methylglyoxal in biological systems is described, together with sample storage, sample processing procedures, and statistical evaluation. Specimen data are presented. Methylglyoxal was assayed by derivatization with 1,2-diamino-4,5-dimethoxybenzene and high-performance liquid chromatography (HPLC) of the resulting quinoxaline, 6,7-dimethoxy-2-methylquinoxaline, with spectrophotometric or fluorescence detection. Derivatization, solid-phase extraction, and HPLC were performed under acid conditions to prevent the spontaneous formation of methylglyoxal from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate during the assay. The limits of detection in the biological matrix were 45 pmol (absorbance detection) and 10 pmol (fluorimetric detection), the recovery was 58%, and the intra- and interbatch coefficients of variance were 7.7 and 30.0%, respectively. The concentration of methylglyoxal in whole blood from normal healthy human individuals was (mean +/- SE, nM) 256 +/- 92 (n = 12) and that from diabetic patients was 479 +/- 49 (n = 55), showing a significant increase in diabetes mellitus (P < 0.01; Mann-Whitney U test). Sample processing under acidic conditions was essential to avoid interferences. Previous estimates of the concentration of methylglyoxal in biological samples require re-evaluation.

The use of ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) to sequester Mg2+ from samples containing ATP at acidic or neutral pH prior to 31P NMR spectroscopic analysis leads to significant broadening of the gamma- and beta-phosphorus resonances of ATP as compared to ATP alone. It was found that the use of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) reduces the broadening of the ATP resonances. At pH 7.0, 30 mM EDTA in the presence of 5 mM ATP and 7 mM Mg2+ leads to a threefold increase in the peak width of the gamma phosphorus of ATP as compared to 5 mM ATP alone. When 30 mM CDTA is used in the place of EDTA, the peak width decreased to about 80% of the peak width of ATP alone. When the experiment is repeated at pH 8.5, both EDTA and CDTA lead to narrow peak widths with no significant difference between the two spectra. At pH 6.0, the use of EDTA leads to a spectrum that is very noisy, with a 10-fold increase in the peak width as compared to ATP in the absence of Mg2+ at this pH, whereas the increase with CDTA is only 50%. These results do not reflect the difference in chelating strength between EDTA and CDTA: The free Mg2+ concentration in the presence of each chelator, as calculated by the computer program given in the Appendix, was nearly equal at each pH. The results, however, reflect a difference in the lability of the metal-ligand bond between EDTA and CDTA.

A highly specific, sensitive, and convenient fluorescence assay for alpha-1,2-mannosidases involved in glycoprotein processing reactions is described. The assay utilizes a coupled enzyme system to determine the amount of free mannose liberated from the disaccharide O-methyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside by the alpha-1,2-mannosidase. The assay was used to determine the substrate specificity of a calcium ion-activated alpha-1,2-mannosidase purified from rabbit liver microsomes. The microsomal mannosidase was specific for hydrolysis of the alpha-1,2 linkage. The mannosyl linkages in alpha-1,3- and alpha-1,6-linked methyl-disaccharides, in methyl-alpha-D-mannopyranoside, and in yeast mannan were hydrolyzed at rates of 2% or less than that noted with the alpha-1,2-linked disaccharide. Mannosidase activity was linear with time and was proportional to enzyme concentration. The Km for the alpha-1,2-linked methyl-disaccharide is 0.5 mM.

β-[U-14C]Alanine can be synthesized in >95% yield from l-[U-14C]aspartic acid using the aspartate 1-decarboxylase of Escherichia coli and converted to d-[1,2,3-14C]pantothenate in a 10–20% yield using the pantothenate synthetase of E. coli. Sufficiently pure preparations of both enzymes are readily obtained.

1-Trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo), a potent toxin toward dopaminergic neurons, readily originates in vitro from the biogenic amine tryptamine and the unnatural aldehyde chloral. For this reason, this heterocycle has been postulated to be formed endogenously in humans after administration of the hypnotic chloral hydrate or after exposure to the industrial solvent trichloroethylene by a spontaneous chemical ring closure reaction. In this paper, we report on the first identification of TaClo in blood samples of patients treated orally with chloral hydrate. Using a specific and sensitive gas chromatographic screening procedure based upon electron-capture and mass-selective detection, TaClo was determined after conversion to its volatile trifluoroacetyl derivative. The identity of TaClo in humans was clearly demonstrated by GC-MS analysis in selected-ion-monitoring mode, by the characteristic chlorine isotopic pattern of the molecular ion.

A high-performance liquid chromatographic method for the fluorometric determination of 1,2,3,4-tetrahydroisoquinoline in rat brain is described. 1,2,3,4-Tetrahydroisoquinoline and 4-phenylpiperidine (internal standard) are isolated by liquid-liquid extraction, and then converted into the corresponding fluorescent derivatives with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, a fluorescence derivatization reagent for amines. The derivatives are separated within 60 min on a reversed-phase column, TSK gel ODS-120T, with isocratic elution, and detected fluorometrically. The detection limit of 1,2,3,4-tetrahydroisoquinoline is 1.0 pmol/g in rat brain (S/N = 3).

A enzyme-linked immunosorbent assay has been developed for tyrosine hydroxylase (TH). The method uses a polyclonal antibody to trap TH, a monoclonal antibody to bind the immobilized TH, a biotinylated, anti-mouse immunoglobulin to bind the monoclonal antibody, and streptavidin covalently coupled to horseradish peroxidase (SA-HRP). The antigen-antibody complex is detected colorometrically following incubation with an HRP substrate. The method detects less than 1 ng (16 fmol) of TH and can be performed in 3 h. The high specificity of the assay is attributed to the use of both polyclonal and monoclonal antibodies, each of which are specific for TH. Data acquisition and reduction is rapid and linked directly to a common desktop computer. Levels of TH protein average 1 ng/microgram protein in striatum and, following treatment with the neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), are decreased to a similar extent as is catalytic activity. In contrast, MPTP did not alter TH homospecific activity. The monoamine oxidase B inhibitor deprenyl blocked both the decrease in activity and the decrease in immunoreactive protein caused by MPTP.

This article describes the employment of a novel p-phenol derivative, 4-(1,2,4-triazol-1-yl)phenol (TRP), as a highly potent signal enhancer of the luminol-hydrogen peroxide (H(2)O(2))-horseradish peroxidase (HRP) chemiluminescence (CL) system. The CL reaction conditions were optimized, and the enhancement characteristics of TRP were compared with those of p-iodophenol (PIP). TRP produced a strong enhancement of the CL with the effect of prolonging the light emission. The developed system was then applied to the determination of H(2)O(2) with immobilized HRP using magnetic beads as a solid support. The linear range for H(2)O(2) was 2.0x10(-6) to 1.0x10(-3) M. The detection limit for H(2)O(2) was 2.0x10(-6) M. The proposed sensor was applied successfully to the determination of H(2)O(2) in rainwater.

Unlike conventional aryl diazonium reagents which are usually reactive at alkaline pH, 3-diazonium-1,2,4-triazole (3-DT) has been found to react with a variety of proteins at acid pH. Using indole, phenol, and imidazole as models of aromatic amino acid side chains, the rate of 3-DT coupling to the indole nucleus has been shown to increase exponentially from <200 m−1 min−1 at pH 6 to > 2400 m−1 min−1 at pH 2.5. Coupling rates with phenol and imidazole show the conventional alkaline reactivity, with no evidence of reaction at acid pH. In parallel experiments using trypsin, 3-DT was found to cause loss in enzymatic activity at acidic as well as alkaline pH. Furthermore, amino acid analysis has demonstrated that the acidic reaction with trypsin resulted in the modification only of tryptophan. Based on the acidic specificity of 3-DT seen in these studies, a scintillation method for determining the tryptophan content of proteins has been developed. Using 3-diazonium-1,2,4-[5-14C]triazole, accurate tryptophan contents for trypsin, chymotrypsin, lysozyme, pepsin, insulin, and fibrinogen have been measured.

Cytosol prepared from small intestine of vitamin D-sufficient rabbits contains a specific high-affinity binding protein for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). This binding protein sediments at 3.0-3.5 S in sucrose density gradients containing 0.3 M KCl. Scatchard analysis using intestinal cytosol demonstrated a Kd of 0.05 nM and a maximum binding capacity of 92 fmol/mg cytosol protein for 1,25(OH)2D3 at 4 degrees C. Competitive binding studies with various metabolites of vitamin D showed a relative binding affinity of this protein for 1,25(OH)2D3 greater than 25-hydroxyvitamin D3 greater than vitamin D3. With 200 micrograms of rabbit intestinal cytosol protein, as little as 1.0-2.5 pg of 1,25(OH)2D3 reproducibly displaced the tracer sterol from the binding protein. Analyses of human plasma 1,25(OH)2D3 content yielded values consistent with published results. The vitamin D-replete rabbit provides a convenient, plentiful, and inexpensive source of binding protein for 1,25(OH)2D3 assays.

A method is described which permits the assay of 1,25-dihydroxyvitamin D in 1 ml of normal human plasma. Samples are extracted with benzene or isopropyl ether. The organic phase is then back-extracted with 0.1 m Na2HPO4, pH 10.5. The final purification step involves high-pressure liquid chromatography on μPorosil with a solvent system of 13% isopropanol in hexane. Recovery through the entire purification procedure averages 76.8%, the purified extract is assayed for 1,25-dihydroxyvitamin D by determining its effect on the resorption of fetal rat bones in vitro. 1,25-Dihydroxyvitamin Da standards in the 4- to 25 pg/ml range gave responses which fitted a logarithmic dose-response curve with an average r2 of 0.986. Repeat assays with normal human plasma gave a coefficient of variation () between experiments of 16.1 ± 0.3%. Normal human serum contains 33.4 ± 6.4 pg/ml () 1,25-dihydroxyvitamin D by this assay. Values averaging less than normal are found in plasmas of patients with renal failure and in patients with pseudohypoparathyroidism. Average values greater than normal were found in patients with hyperparathyroidism. 1,25-Dihydroxyvitamin D was not detectable in plasma of anephric patients.

A mathematical analysis of the kinetics of the hormone-receptor interaction was applied to the 1,25-dihydroxycholecalciferol-intestinal receptor system. The exact analytical solution and the numerical integration of the kinetic equation were installed in a Statistical Analysis System (SAS) computer program to estimate the rate constants of the reaction. Estimates of the parameters obtained by these two methods are similar, demonstrating that the numerical integration can be combined with the nonlinear regression procedure for least-squares parameter fitting using a simple SAS program. This enables estimation of kinetics rate constants when the kinetic equation cannot be solved analytically. The ratio of the rate constants (ka/kd) found by the nonlinear procedure is close to the independently determined equilibrium (Scatchard) constant in the nonlinear analysis.