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The present work reports the identification and characterization of a contaminant, 2-(2'-(1,5-naphthyridinyl))-1,3-indanedione (1',5'-naphthyridinophthalone, 1,5NP), in the color additive DC Yellow No. 10 (U.S.-certifiable form of Quinoline Yellow), together with its quantification in batches of the color additive certified by the U.S. Food and Drug Administration (USFDA). The impurity, which is a compound not previously reported in the literature, was synthesised and characterised for use as a reference material. Test portions from 26 certified batches of DC Yellow No. 10 submitted to USFDA by four domestic and four foreign manufacturers were analyzed for 1,5NP using high-performance liquid chromatography. The results revealed a wide range of 1,5NP levels across batches, with 18 (69.2%) of the test portions containing amounts from 0.32 to 169.94 µg g

The nature of the inhibition of thiol proteases by a new class of mechanism-based inhibitors, 1,5-diacylcarbohydrazides, is described. These potent, time-dependent, active-site spanning inhibitors include compounds that are selective for cathepsin K, a cysteine protease unique to osteoclasts. The 1,5-diacylcarbohydrazides are slow substrates for members of the papain superfamily with inhibition resulting from slow enzyme decarbamylation. Enzyme-catalyzed hydrolysis of 2,2'-N, N'-bis(benzyloxycarbonyl)-L- leucinylcarbohydrazide is accompanied by formation of a hydrazide-containing product and a carbamyl-enzyme intermediate that is sufficiently stable to be observed by mass spectrometry and NMR. Stopped-flow studies yield a saturation limited value of 43 s(-)(1) for the rate of cathepsin K acylation by 2,2'N, N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide. Inhibition potency varies among proteases tested as reflected by 2-3 orders of magnitude differences in K(i) and K(obs)/I, but all eventually form the same stable covalent intermediate. Reactivation rates are equivalent for all enzymes tested (1 x 10(-)(4) s(-)(1)), indicating hydrolysis of a common carbamyl-enzyme form. NMR spectroscopic studies with cathepsin K and 2,2'-N,N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide provide evidence of inhibitor cleavage to generate a covalent carbamyl-enzyme intermediate rather than a tetrahedral complex. The product Cbz-leu-hydrazide does not appear enzyme-bound after cleavage in the NMR spectra, suggesting that the stable inhibited form of the enzyme is the thioester complex. 1, 5-diacylcarbohydrazides represent a new class of unreactive cysteine protease inhibitors that share a common mechanism of action across members of the papain superfamily. Both S and S' subsite interactions are exploited in achieving high selectivity and potency.

Cathepsin K (EC 3.4.22.38) is a cysteine protease of the papain superfamily which is selectively expressed within the osteoclast. Several lines of evidence have pointed to the fact that this protease may play an important role in the degradation of the bone matrix. Potent and selective inhibitors of cathepsin K could be important therapeutic agents for the control of excessive bone resorption. Recently a series of peptide aldehydes have been shown to be potent inhibitors of cathepsin K. In an effort to design more selective and metabolically stable inhibitors of cathepsin K, a series of electronically attenuated alkoxymethylketones and thiomethylketones inhibitors have been synthesized. The X-ray co-crystal structure of one of these analogues in complex with cathepsin K shows the inhibitor binding in the primed side of the enzyme active site with a covalent interaction between the active site cysteine 25 and the carbonyl carbon of the inhibitor. ©

Growth and survival of Epstein-Barr virus (EBV)-immortalized B lymphocytes cultured at low cell densities require autocrine soluble factors. In this study, we have purified a low molecular weight autocrine soluble factor that promotes growth of EBV-immortalized B cells in serum-free conditions and identified it as lactic acid (LA). Synthetic LA stimulated growth in EBV-immortalized B cells at 1-10 mM, a concentration of LA measured in the culture supernatant of EBV-immortalized cell lines. LA alone was found to account for greater than 70% of the autocrine growth factor activity in serum-free supernatants of EBV-immortalized B cells. Aminooxyacetate, a glutamate-oxaloacetate transaminase inhibitor, specifically inhibited B-cell growth induced by LA, suggesting that this process requires mitochondrial-cytosol transfers. Thus, LA is an autocrine stimulatory molecule that in serum-free conditions is essential for the continuous proliferation of EBV-immortalized B cells. This represents an unexpected function for LA.

A versatile probe-mounted interface for supercritical fluid chromatography Fourier transform mass spectrometry has been designed, constructed, and evaluated. The interface is fully contained within a 40 in. long stainless steel tube and provides for accurate transfer line temperature control and separate control of a noninductively wound probe tip heater. Functional characteristics of the new SFC/FTMS probe interface are demonstrated by analyses of cholesterol and a polyaromatic hydrocarbon mixture. Detection limits of approximately 100 pg are demonstrated for aromatic hydrocarbons

Bioassay-guided fractionation of an active methyl ethyl ketone extract of Hardwickia binata, using an assay sensitive to DNA polymerase beta inhibition, resulted in the isolation of a potent inhibitor. This proved to be a novel diterpenoid, which has been named harbinatic acid (1). The structure of 1 was established as 3alpha-O-trans-p-coumaroyl-7-labden-15-oic acid from spectroscopic analysis and by comparison with the published data for a structurally related compound. Compound 1 strongly inhibited calf thymus DNA polymerase beta, with an IC(50) value of 2.9 microM.

Using binding models which were based on the X-ray crystal structure of an amino acid-based active site-spanning inhibitor complexed with cathepsin K, Cbz-leucine mimics have been developed, leading ultimately to the design of a potent cathepsin K inhibitor free of amino acid components. These mimics, which consist of alpha-substituted biphenylacetyl groups in place of Cbz-leucine moieties, effectively mimic all aspects of the Cbz-leucine moieties which are important for inhibitor binding. The predicted directions of binding for the inhibitors were confirmed by mass spectral analysis of their complexes with cathepsin K, which gave results consistent with acylation of the enzyme and loss of the acylhydrazine portion of the inhibitor which binds on the S' side of the active site. The binding models were found to be very predictive of relative inhibitor potency as well as direction of inhibitor binding. These results strengthen the validity of a strategy involving iterative cycles of structure-based design and inhibitor synthesis and evaluation for the discovery of non-peptide inhibitors.

The in vitro activation of the recombinant purified human cathepsin K (EC 3.4.22.38) was examined by mutagenesis. Cathepsin K was expressed as a secreted proenzyme using baculovirus-infected Sf21 insect cells. Spontaneous in vitro activation of procathepsin K occurred at pH 4 and was catalyzed by exogenous mature cathepsin K. Three intermediates were identified as resulting from cleavages after Glu19, Ser98, and Glu110. The mature enzyme was composed of mixture of enzymes with N termini of Gly113, Arg114, and Ala115 with varying ratios depending on the preparation. Molecular weight determinations were consistent with the absence of carbohydrate in the mature protein, while electrospray mass spectroscopy indicated that six of the eight cysteine residues were in disulfide linkage, and that the protein had Met329 as the C-terminal residue. A mutant was constructed in which the active site Cys139 was changed to Ser. [Ser139,Ala163]Procathepsin K (containing mutation C139S,S163A) failed to spontaneously process and was only partially processed in the presence of 1% exogenous wild-type mature cathepsin K forming intermediates, which were identical to those observed in the activation of wild-type. [Ser139,Ala163]Procathepsin K could be fully processed to mature enzyme by including one equivalent of wild-type procathepsin K in the activation mixture. These results indicated that in vitro activation of the procathepsin K was an autocatalytic process.

It has been shown that the pituitary contains a cytotropic factor (CTF) that stimulates the secretion of catecholamines by dopaminergic neurons of the hypothalamus. In the present study, CTF was purified from rat pituitaries and found by means of mass spectrometric analysis to be adenosine. This finding was corroborated by the observations that CTF behaves identically to adenosine when subjected to liquid chromatography, is inactivated and converted to inosine by adenosine deaminase, and is qualitatively and quantitatively indistinguishable from adenosine in its biological activity. It is concluded that pituitary adenosine is a trophic factor for hypothalamic dopaminergic neurons.

Use of infusion methods rather than high-performance liquid chromatography allowed us to confirm the observation that solutions of propionic acid-isopropanol restore sensitivity lost due to trifluoroacetic acid in electrospray mass spectra of basic substances, particularly peptides. In this work, when propionic acid-isopropanol was used, we detected an abundant ion with m/z 622 that shifted to m/z 538 when we substituted acetic acid-methanol for the propionic acid-isopropanol. Via accurate mass measurement and tandem mass spectrometry the origin of the ion was identified as the complex Fe3O(O2CR)6(L)0–3, where L is one of several ligands from solvent or water. The grounding arrangement of the Finnigan TSQ-700 electrospray source produces electrolytic currents that may accentuate the abundance of this complex and specifically produces observable gas bubbles that adversely affect the spray stability.

An efficient ion transport system that interfaces external ion sources with a commercial dual-cell Fourier transform mass spectrometry (FTMS) system so as to retain maximum experimental flexibility has been constructed. Electrostatic lenses were used for ion transfer with potentials less than 200 V to preclude discharges. Spectra were recorded by thermal ionization and by electrospray ionization. Other high pressure ionization methods can be easily added to the external ion source chamber, making this a general solution for ion transport into an FTMS system. The efficiency of ion transfer was measured to be approximately 30%. A pressure ratio of 105 between the external ion source chamber and the second cell has been demonstrated. The system incorporates a computer-controlled gate valve to isolate the cell regions from the external ion source chamber, permitting optimal conditions for ion injection and accumulation, and then after closing the valve, recording spectra at low pressure with high resolution. Spectra of Gramicidin S (resolution 90,000 at m/z 1164), aprotinin (resolution 410,000 at m/z 1304), and horse heart cytochrome c (resolution 50,000 at m/z 1546) are shown.

Photodissociation (PD) and surface-induced dissociation (SID) are compared for structural analysis of several nonvolatile compounds analyzed by laser desorption Fourier transform mass spectrometry (LD/FTMS). SID and PD of a porphyrin and two metalloporphyrins were investigated using a variety of experimental conditions. Optimum structural information is obtained from PD when parent ions are irradiated for relatively long times (10-30 s) using 575-nm radiation and short times (0.5-1 s) using 308- or 388-nm radiation. Shorter irradiation times in the visible region resulted in less efficient production of structurally significant product ions, while longer times in the ultraviolet region produced more nonspecific fragment ions, apparently at the expense of more structurally significant fragment ions. SID conversion efficiencies for the porphyrins are estimated for collision energies from 25 to 360 eV, with maximum conversion efficiency found using 62- and 115-eV collision energies for the two porphyrins studied. Results from a concurrent study on the combined use of PD and SID for MS/MS/MS are discussed in the context of these results. The MS3 ion spectra generated by the two dissociation techniques differ more significantly than MS2 product ion spectra. These data suggest some general guidelines for MSn studies of nonvolatile compounds analyzed by LD/FTMS, employing PD and SID for ion activation.

A detailed procedure for performing surface-induced dissociation (SID) of ions in a dual-cell Fourier transform mass spectrometer is described. It is shown that the technique is applicable to both electron ionization and laser desorption measurements. SID spectra of perfluorotri-n-butylamine, anthracene, (5,10,15,20-tetraphenyl-21H,23H-prophinato)-iron(III) chloride, and [5,10,15,20-tetrakis(2,6-dibromo-phenyl)-21H,23H-prophina to]iron(III) chloride are presented. Conversion efficiencies of molecular ions between 1% and 30% are obtained. It is concluded the method holds promise for dissociation of high mass laser-desorbed ions.

The pressure requirements for chemical ionization g.c./F.t.m.s. which restrict mass resolution and accuracy are overcome through use of a pulsed valve that provides momentary reagent gas pressures. For alternate electron impact (EI)/chemical ionization (c.i.) g.c./F.t.m.s., similar resolution for both e.i. and c.i. data is demonstrated. The efficiency of chemical ionization with the pulsed valve is similar to static high pressure c.i. measurements of several model compounds. Results from the analysis of peppermint oil and a fuel additive illustrate the potential information available from a single g.c./F.t.m.s. experiment.

Previous Fourier transform mass spectrometry (FTMS) measurements have commonly utilized frequency swept excitation. This communication describes the first demonstration of random-noise excitation for FTMS employing a simple noise excitation source. Characteristics of the source are discussed and a random excitation spectrum is compared with the corresponding frequency sweep excitation spectrum.

A differentially pumped external secondary ion source for Fourier transform mass spectrometry is described. Installation does not interfere with other experiments such as laser desorption or photodissociation. Spectra of cesium iodide clusters (with ions up to m/z 8187), polyethylene glycol 1000, and histidine from both glycerol and dithiothreitol-dithioerythritol matrices are reported. The ion with m/z 156 from histidine was recorded with mass resolution of 160,000.

La technique decrite permet l'identification des differents composes dans des melanges complexes