Potential of porphyrins as chromogenic reagents for determining metals in capillary electrophoresis.

Although capillary electrophoresis (CE) with photometric detection is a well-established technique for the determination of various inorganic ions, its limited sensitivity has hindered greater development in this area. In this work, we used a mixture of metals consisting of Co(II), Ni(II), Zn(II) and Mn(II) to demonstrate that the sensitivity of CE with ultraviolet-visible (UV-vis) detection can be improved by using chromogenic reagents such as porphyrins. To this end, the metals were reacted with 5,10,15,20-tetrakis(4-sulphophenyl)-porphine dodecahydrate (TPPS(4)) to obtain their respective porphyrinato complexes, which were then separated by CE with a citrate buffer and detected at 410 nm. The ensuing electrophoretic method has a limit of detection (LOD) of 3 x 10(-6) M (180 microg L(-1)) for Co(II), 2 x 10(-10) M (0.012 microg L(-1)) for Ni(II), 4 x 10(-6) M (260 microg L(-1)) for Zn(II) and 4 x 10(-9) M (0.219 microg L(-1)) for Mn(II). The method is a highly promising choice for the ultratrace determination of Ni(II) and Mn(II).

Comparison of off- and in-line solid-phase extraction for enhancing sensitivity in capillary electrophoresis using ochratoxin as a model compound.

This paper proposes and compares two approaches based on off- and in-line solid-phase extraction (SPE), intended to enhance sensitivity in capillary electrophoresis with ultraviolet detection (CE-UV) using as a model the determination of ochratoxin A (OA) in river water samples. In the off-line SPE mode, the reversed-phase sorbent (octadecilsylane, C(18)) selectively retains the target analyte (OA) and allows large volumes of the sample (70 mL) to be introduced and subsequently eluted in a small volume (0.1 mL) of an appropriate solution. In the in-line SPE mode, a custom-made microcartridge is inserted near the inlet of the capillary, which is filled with the same C(18) sorbent. This solid phase selectively retains OA present in a sample volume as low as approximately 640 microL for subsequent elution with ca. 135 nL of an appropriate eluent. The limit of detection (LOD) obtained with the in-line SPE method was 1 ng L(-1), which is 3 orders of magnitude lower than that obtained with CE-UV and roughly 1 order lower than that provided by the off-line SPE-CE-UV method.

Direct determination of chlorophenols present in liquid samples by using a supported liquid membrane coupled in-line with capillary electrophoresis equipment.

Actually there is a great trend on the development of effective analytical methods for monitoring trace levels of various phenols which can indicate, among others compounds, the water quality. A simple, inexpensive supported liquid membrane (SLM) device was used in combination with commercially available capillary electrophoresis (CE) equipment for the direct determination of chlorophenols in surface water samples. The manifold was used simultaneously to extract and preconcentrate the analytes from liquid samples. In the extraction set-up, the donor phase (4 mL) was placed in the CE vial, where a micro-membrane extraction unit (MMEU) accommodating the acceptor phase (100 microL) in its lumen was immersed. The supported liquid membrane was constructed by impregnating a porous Fluoropore Teflon (PTFE) membrane with a water-immiscible organic solvent (dihexyl ether). The extraction process was optimized with regard to the pH of the donor and acceptor phases, membrane liquid, extraction time and voltage applied to the inlet or outlet vial during extraction. The chlorinated phenols pentachlorophenol (PCP), 2,3,6 trichlorophenol (TCP) and 2,6 dichlorophenol (DCP) were thus efficiently separated by CE, using tris(hydroxymethyl)aminomethane (Tris) and an NaH(2)PO(4) solution containing 1% (v/v) methanol at pH 10.5 as running buffer.

A new myosin fragment: visualization of the regulatory domain.

Many of the functional domains of the myosin molecule have been defined by the use of proteolytic enzymes. Major fragments that retain enzymatic or assembly properties have been prepared by cleavage in the rod to form heavy meromyosin (HMM) and light meromyosin (LMM) or at the head-rod junction to form S-1 and rod. Limited tryptic digestion of vertebrate skeletal myosin S-1 indicates that the head contains three major regions: an amino-terminal nucleotide binding domain of molecular weight (MW) 25,000, a central domain of MW 50,000 and a carboxyl domain MW 20,000; the latter two are both able to bind to actin. Tryptic digestion of scallop S-1 has also been used to isolate a head fragment MW 14,000 associated with both types of scallop light chains. Here we report that myosin from vertebrate (chicken and rabbit skeletal) and molluscan (scallop adductor) striated muscles is cleaved in an unusual way with an enzyme from Pseudomonas aeruginosa. This bacterial protease (designated Ps-1) does not cleave myosin at the head-rod junction or in the rod; instead, Ps-1 splits the myosin heavy chain within the head, yielding a complete rod joined to the 20,000-MW head domains. The scallop regulatory and essential light chains remain associated with this fragment. We examined this new fragment by electron microscopy; the rods bear two 'nubs' about 100 A long, which appear to correspond morphologically to the neck region of the myosin molecule.

Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases.

A protein capable of inhibiting trypsin and other pancreatic proteases has been purified to homogeneity from Escherichia coli by conventional procedures and affinity chromatography. It is stable for at least 30 min at 100 degrees C and pH 1.0, but it is inactivated by digestion with pepsin. The inhibitor has an apparent molecular weight of 38,000 as determined by gel filtration and must be a homodimer since it contains a single 18,000-dalton subunit upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor has an isoelectric point of 6.1. One dimeric molecule of the inhibitor can bind two trypsin molecules to form a mixed tetrameric complex, in which trypsin molecules are completely inhibited. The inhibitor is not digested by the trypsin. When N-benzoyl-DL-arginine-p-nitroanilide was used as a trypsin substrate, half-maximal inhibition was observed at 22 nM. This protein also inhibits chymotrypsin, pancreatic elastase, rat mast cell chymase, and human serosal urokinase, but it does not inhibit human pulmonary tryptase, kallikrein, papain, pepsin, Staphylococcus aureus V8 protease, subtilisin, and thermolysin. Surprisingly, it did not inhibit any of the eight soluble endoproteases recently isolated from E. coli (i.e. proteases Do, Re, Mi, Fa, So, La, Ci, and Pi) nor the chymotrypsin-like (protease I) and trypsin-like (protease II) esterases in E. coli. The inhibitor is localized to the periplasmic space and its level did not change with different growth media or stages of cell growth. The physiological function of this E. coli trypsin inhibitor is unknown. We suggest that E. coli trypsin inhibitor be named "Ecotin."

The binding properties of human complement component C1q. Interaction with mucopolysaccharides.

Quantitative measurements have been made of the interaction of human complement subcomponent C1q with mucopolysaccharides. The binding of C1q to heparin was quantitatively examined by utilizing an assay that employs a 125I-labeled low molecular weight heparin glycosaminoglycan (LMW-Hep) (Mr = 8500). Two classes of binding sites were detected. The first class of sites bound 2.02 mol of LMW-Hep/mol of C1q with a Kd of 76.6 nM. The second class of sites complexes with 12 mol of LMW-Hep with a Kd of 1.01 microM. The higher affinity-binding site for LMW-Hep could be assigned to the collagenous region of C1q (C1q-c); 2.2 mol of 125I-LMW-Hep were bound/mol of purified isolated C1q-c with a Kd = 381 nM. In contrast, the isolated C1q globular region did not bind to 125I-LMW-Hep. The binding of LMW-Hep to C1q and the C1q-c region was confirmed by fluorescence polarization experiments; C1q and C1q-c bound 2.3 and 2.02 mol of fluorescamine-labeled LMW-Hep/mol of protein, respectively. A variety of mucopolysaccharides were able to inhibit interaction of C1q with 125I-LMW-Hep, the most effective being heparan sulfate and dermatan sulfate. LMW-Hep (2.5 nM) inhibited the ability of C1q (0.5 nM) to recombine with C1r (1.4 nM) and C1s (1.6 nM) to form hemolytically active C1. At 250 mM, LMW-Hep inhibited the hemolytic activity of reconstituted C1. The ability of mucopolysaccharides to interact with purified C1q suggests a role for such molecules in the regulation of the first component of complement.

Fibronectin binds to the C1q component of complement.

Fibronectin immobilized to plastic tubes binds soluble C1q with a Kd of 82 +/- 2.6 nM. The binding of fibronectin to C1q is relatively insensitive to pH but is sensitive to ionic conditions. C1q covalently bound to Sepharose selectively binds cellular fibronectin produced by a hamster fibroblast cell line. The globular head regions of C1q have no effect on the binding of C1q to fibronectin but the collagenous tails of C1q interfere competitively with a Ki of 59 nM. We conclude that fibronectin binds C1q via its collagen-like tail region and thus the process resembles the binding of fibronectin to gelatin. This is further emphasized by our observation that gelatin binds to fibronectin immobilized on plastic tubes with a Kd of 131 nM. Because fibronectin stimulates endocytosis in several systems and promotes the clearance of particulate material from the circulation, these results suggest the possibility that fibronectin could function in the clearance of C1q-coated material such as immune complexes or cellular debris.

Photoaffinity inhibition of rat liver NAD(P)H dehydrogenase by 3-(alpha-acetonyl-p-azidobenzyl)-4-hydroxycoumarin.

NAD(P)H dehydrogenase was purified in four steps from a homogenate of rat liver. The final step was affinity chromatography on Sepharose coupled to 3,3'-(m-hydroxybenzylidene)bis(4-hydroxycoumarin). The purified enzyme was inhibited competitively with respect to NADH by 3-(alpha-acetonyl-p-nitrobenzyl)-4-hydroxycoumarin (acenocoumarin) (Ki = 1.7 microM). The acenocoumarin was converted into an azide which was used to photoaffinity inhibit the enzyme. Following photolysis in the presence of the azide, the enzyme was inactivated in proportion to the concentration of azide present during irradiation. A maximum of 35-40% inhibition could be achieved by a single irradiation at 254 nm for 1.5 min. This inhibition was noncompetitive with respect to NADH. The inactivation was shown to be specific as acenocoumarin afforded complete protection against inactivation, irradiation was required to achieve inactivation, and the enzyme was unaffected by irradiation alone.