Analysis of porphyrin carboxylic acids in biological fluids by high-performance liquid chromatography

A method for the rapid and sensitive fluorometric analysis of porphyrin carboxylic acids by reverse-phase high-performance liquid chromatography is described. Separation of free porphyrin carboxylic acids was carried out with a microparticulate octadecylsilane column with elution by a gradient of methanol in phosphate buffer containing tetrabutylammonium hydroxide. Separation and quantitation of di-, tri-, tetra-, penta-, hexa-, hepta-, and octacar-boxylic porphyrins was achieved within 25 min at picomolar concentrations. The method is also capable of separating the type I and type III isomers of tetracarboxylic through hexacarboxylic porphyrins. By using a stopped flow technique, one can record fluorescence excitation and emission spectra of porphyrin carboxylic acids. This method is directly applicable to biological fluids such as urine, plasma, red cell lysates, or medium or extracts from cell culture.     

Spectrophotometric determination of myoglobin in cardiac and skeletal muscle: separation from hemoglobin by subunit-exchange chromatography.

Myoglobin is extracted from muscle and separated from blood hemoglobin by subunit-exchange chromatography on a column of Sepharose 4B to which hemoglobin α-β subunits are linked covalently. Hemoglobin is retained on the column. Myoglobin in the effluent is determined spectrophotometrically as ferrous myoglobin or as carbon monoxide ferrous myoglobin. The method is applicable to cardiac, smooth, or skeletal muscle from mammals, reptiles, birds, and teleost fish, but failed with the one amphibian and the one shark tested.     

A rapid assay for prolyl 3-hydroxylase activity.

An assay is reported for prolyl 3-hydroxylase activity. The method is based on the release of tritiated water (THO) during 3-hydroxylation of a 2,3-T-l-proline-labeled (T = tritium) polypeptide substrate in which all prolyl residues recognized by prolyl 4-hydroxylase have been converted to 4-hydroxyprolyl residues. The formation of THO was essentially linear with enzyme concentration and time, and the K_m for the polypeptide substrate was about 3.4 × 10^?8m. A linear correlation was found between THO release and the synthesis of 3-hydroxyproline, the latter being analyzed by amino acid analyzer. The assay is simple, rapid, sensitive, and reproducible, and it is specific even in tissue samples containing a large excess of prolyl 4-hydroxylase activity.     

Histidine regulation in Salmonella typhimurium: XVI. A sensitive radiochemical assay for histidinol dehydrogenase

A sensitive radiochemical assay for measurement of histidinol dehydrogenase is presented. The method is based upon separation of the product of the reaction. [¹⁴C]histidine, from the substrate, [¹⁴C]histidinol, on small Dowex 50 columns. The assay can be performed on cell extracts or on toluenized cells and is approximately 100 times more sensitive than previously reported assays for this enzyme.[¹⁴C]histidinol is obtained in high yields through conversion of uniformly labeled ¹⁴C-glucose by a strain of Salmonella typhimurium derepressed for the histidine operon and blocked at the histidinol dehydrogenase step. Accumulated [¹⁴C]histidinol is purified from the culture supernatant by ion-exchange chromatography.This sensitive assay has facilitated measurement of reduced levels of histidine operon expression in promoter mutants, and has been adapted for study of histidine operon regulation in a cell free protein synthesizing system.     

Fibrin membrane endowed with biological function. VII. An approach to an artificial liver; conversion of ammonia to urea in vitro

As an “artificial liver” for the conversion of ammonia to urea, a group of enzymes in ornithine cycle together with carbamyl phosphate synthetase I and inorganic pyrophosphatase were embedded in a single fibrin membrane. The immobilized enzyme system thus prepared had an ability to convert ammonia to urea not only in a buffer solution but also in human plasma.     

An immunocytotoxicity assay for neural cells in monolayer cultures

The immunological analysis of cell surface constituents which may characterize neuronal and glial populations, though still in its infancy, will greatly facilitate the investigation of several important problems in neurobiology. One critical component of such analyses is the way by which a given antiserum can be shown to be active on, and possibly selective for neurons and glial cells from normal neural tissues. This report describes the use of monolayer cultures of normal neural cells for recognition and quantitative titration of antisera directed against them. Sera were collected from rabbits immunized with chick embryo spinal cord cell susptnsions, and found to be reactive to the same cells in the initial cell dissociate as well as in subsequent monolayer cultures of different in vitro ages. A monolayer assay procedure was developed, which (i) uses small numbers of cells and small volumes of immune reagents, with the possibility of further scaling down; (ii) applies equally to cultures using different substrata; (iii) permits differential counts of morphologically different cultured cells; (iv) allows to recognize cytological damage imposed by the immune serum in the presence, though not in the absence, of complement; and (v) quantitatively titrates the immune activity with 10- to 20-fold higher sensitivity than other titration procedures. While the study was not intended to investigate the possible specificities of the new antisera, it provided the unexpected observation that non-neuronal cells in these spinal cell cultures were considerably less sensitive than neurons to the complement-dependent action of the antisera.     

Lactose biosynthesis: A sensitive radiochemical assay

A method is presented for the determination of lactose biosynthesis from labeled glucose, galactose, or other precursors based upon the addition of samples of the reaction mixture (after removal of the tissue or biosynthetic enzymes) to each of two strains of Escherichia coli. While both strains can metabolize glucose and galactose, only one is able to hydrolyze lactose. The sugars are converted by the bacteria largely to cell material and carbon dioxide. The difference between the residual, nonvolatile, soluble radioactivity in the medium from the two bacterial cultures represents the lactose unused by the strain unable to hydrolyze it.     

Improved technique for accurate and convenient assay of biological reactions liberatingCO2

Use of a convenient and inexpensive apparatus for trapping¹⁴CO₂ from biological reactions in small volumes is described. Alternative scintillants for estimating¹⁴CO₂ trapped in KOH are compared, toluene methoxyethanol (2:1) based scintillants and Instagel (TM-Packard) were efficient and more stable than the dioxane/Cab-O-Sil or toluene/Triton X-100 mixtures usually used. The usefulness of the technique is illustrated with microassays of OMP decarboxylase (EC 4.1.1.23), OMP pyrophosphorylase (EC 2.4.2.10) in the fission yeastSchizosaccharomyces pombe.     

An improved rapid assay of glycogen synthase

As an alternative to rapid filtration washing the glycogen free of any unreacted UDP-[¹⁴C]-glucose by ascending chromatography (ethanol:water, 2:1) can be used. This technique also makes the filter paper assay of glycogen synthase much faster: The samples are ready for liquid scintillation counting in 30 min. Among the other advantages offered by this procedure, we should also mention that blanks are very low, large volumes of ethanol can be saved, and the unreacted UDP-[¹⁴C]glucose can be recovered by elution and recycled (it migrates with the front of the solvent).     

Protein kinase assay on isoelectric focusing gels: evaluation of its quantitative potentials.

The conditions of the Hirsch-Rosen assay (1974, Anal. Biochem.60, 389–394) for protein kinase activity oncomplete isoelectric focusing gels have been analyzed and further developed with the consequence that the test can be easily adapted to other protein kinases in a quantitative manner. Special attention was given (i) to the breakdown of the pH gradient in relation to gel size and Ampholine concentration in order to achieve optimal pH ranges for the assay, (ii) to the introduction of histone as a substrate besides protamine, and (iii) to the distribution of the particular substrates and products throughout the gel. The results of the protein kinase assay on gels were shown to be linear for at least 1 h, and to be dependent on the amount of ATP and on the amount of protein kinase applied, thereby fulfilling the requirements necessary to yield quantitative data.     

Superoxide dismutase assay using alkaline dimethylsulfoxide as superoxide anion-generating system

Alkaline dimethylsulfoxide as a superoxide anion-generating system in association with cytochrome c as a superoxide anion-indicating scavenger has been used to develop a new assay for superoxide dismutase. The assay is sensitive (one unit of enzymatic activity is provided by 110 ng of purified copper-containing superoxide dismutase) and highly specific. The nature of this system prevents the usual interferences and its simplicity allows for multiple, rapid measurements of superoxide dismutase activity in biological preparations using either normal or automated procedures.     

Insulin polymorphism: some physical and biological properties of rat insulins

Although rat insulins I and II show no significant differences in their biological activities and receptor binding on isolated fat cells, X-ray studies and circular dichroism indicate that they have differences in their structures. Rat insulin II forms zinc insulin hexamers in an identical manner to bovine insulin, but insulin I, which has a unique proline substitution at B9, forms hexamers less easily. Rat insulin I can form zinc insulin hexamers given higher zinc concentrations, as indicated by the formation of rhombohedral 2Zn insulin crystals. On the other hand, rat insulin II forms cubic crystals of space group $\text{P4}{}_2\text{32}\text{with}\text{a = 67}\text{A}\text{?}$ under similar conditions. Model building indicates that these crystals contain a tetrahedral arrangement of zinc hexamers. They have a higher solvent content and are less stable than rhombohedral insulin crystals. The relation of these observations to the rat insulin storage granules and the importance of polymorphism to the physiology and evolution of insulin are discussed.     

Rabbit-liver phosphorylase: improvement of the purification procedure and assay of the inactive b form

Continuous electrophoresis buffers are described for polyacrylamide gels at pH values ranging from 3.8 to 10.2. The buffers consist of an acidic and a basic component with pK values near the pH of the buffer. The pH is maintained to within 0.5 pH unit in the electrode compartments during prolonged electrophoresis. Some proteins produce clear bands on gels with each of the 10 buffers. The buffers provide an expansion of the pH range of gel electrophoresis and are likely to be useful in the detection of genetic variation in proteins and in other applications.     

A rapid,sensitive assay for starch phosphorylase and ADPglucose pyrophosphorylase

Starch phosphorylase (EC 2.4.1.1) and ADPglucose pyrophosphorylase (EC 2.7.7.27) activities can be measured very accurately and quickly using a recording pH meter. Activities less than 0.28 nmol/min are readily measured.     

GABA uptake inhibitors produce a greater antinociceptive response in the mouse tail-immersion assay than other types of GABAergic drugs

Antinociception produced by the GABA uptake inhibitors d,l- SKF-89976A and SKF-100330A was characterized and compared to that produced by other types of GABAergic drugs. Using the mouse tail-immersion assay it was found that the antinociception produced by the uptake inhibitors was antagonized by scopolamine, a cholinergic muscarinic receptor antagonist. However, neither SKF compound demonstrated any significant affinity for muscarinic receptor binding sites suggesting that they are not direct-acting cholinomimetics. In vitro uptake experiments revealed that the SKF compounds selectively inhibit GABA transport, having no effect on the accumulation of aspartic acid, glutamic acid, beta-alanine or glycine. Moreover, antinociception and GABA uptake inhibition were stereoselective for SKF-89976A, with the d-isomer being more active in both tests. When comparing antinociceptive responses at maximally effective doses it was also found that the SKF compounds were substantially more efficacious than direct-acting GABA receptor agonists or a GABA transaminase inhibitor. These data suggest that uptake inhibitors may be facilitating GABA transmission in a system that is less affected by other types of GABAergic compounds.     

Synthesis and biological properties of ovine corticotropin-releasing factor (CRF)

The 41-residue sequence of recently identified ovine corticotropin-releasing factor (CRF) was assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptide was purified by gel filtration and reverse-phase, medium pressure, followed by high-performance liquid chromatography (HPLC). In addition to the usual criteria, the homogeneity of the final material, obtained in 7% yield, was assessed by the isolation and examination of cyanogen bromide cleavage and tryptic digestion fragments by HPLC and amino acid analysis. The synthetic 41 amino acid CRF stimulated the release of corticotropin (ACTH) in three in vitro systems: isolated rat pituitary quarters, monolayer cultures of dispersed pituitary cells, and superfused pituitary cells on a column, the responses being related to the log-dose of CRF in the range of 0.05-125 ng/ml. The synthetic peptide also augmented in vivo release of ACTH in rats pretreated with chlorpromazine, morphine, and Nembutal, as assessed by the measurement of serum corticosterone. The data indicates chemical purity and high biological activity of synthetic material.     

A simplified assay of furosemide in plasma and urine by high-pressure liquid chromatography

A simplified high-pressure liquid chromatographic method for determination of furose-mide in plasma and urine has been developed using a fluorometric detector directly coupled to the column effluent. The method includes an ether extraction from acidified biologic samples. The mobile phase used for chromatography on a reversed-phase column (C₁₈ hydro-carbon permanently bonded to silica particles) is sufficiently acidic to induce fluorescence of furosemide. The methylester of furosemide is employed as an internal standard. The sensitivity is 0.1 and 0.25 μg per ml plasma and urine, respectively. The applicability to pharmacokinetic studies of furosemide is shown.     

(Na+,K+)-ATPase: a new assay of Na+-ATPase reveals covert anti-pump antibodies

A new assay is described for rat (Na⁺,K⁺)-ATPase [EC 3.6.1.3] prepared from renal medullary or crude liver membranes. With ATP at 1 μm, initial rates of ouabain-sensitive decreases in substrate concentrations are followed by measuring diminished ATP-driven luciferin-luciferase light production. Under these conditions, using highly purified enzyme preparations, Na⁺ and K⁺ ions stimulate and inhibit initial ATP hydrolysis rates, respectively. Therefore, it is likely that the assay measures Na⁺-ATPase partial reactions of the pump. A monospecific polyclonal rabbit anti-rat pump antiserum blocks Na⁺-dependent ATPase measured with the luciferase-linked ATPase assay, whereas conventional assays of purified pump activity at 3.0 mm ATP fail to reveal immunochemical blockade.     

Spectrophotometric assay of NADase-catalyzed reactions

A spectrophotometric method for the assay of NADase-catalyzed reactions was developed. The assay consisted of monitoring the decrease in absorbance at 275 nm accompanying the enzyme-catalyzed hydrolysis of ?-NAD. A millimolar extinction coefficient of 0.89 at 275 nm was determined for the hydrolysis of the nicotinamide-ribosidic bond of ?-NAD. Under assay conditions the assay was shown to be linear up to 50% completion. A linear relationship between the rate of ?-NAD hydrolyzed and the amount of NADase added was observed. The K_m and V_max values for Bungarus fasciatus venom NADase-catalyzed hydrolysis of ?-NAD were determined spectrophotometrically and were shown to be the same as those determined by other analytical methods.     

Quantitation of chlordiazepoxide and its metabolites in biological fluids by thin-layer chromatography

Chlordiazepoxide and its 4 major metabolites were assayed after separation by thin-layer chromatography following extraction from biological fluids. The compounds become intensely fluorescent in the presence of red, fuming nitric acid. The resulting compounds are quantitated with a spectrodensitometer with a fluorescent attachment. The sensitivity varies between 0.05 and 0.1 μg. The coefficient of variation is 1.4% for assays in urine and 6.4% in serum.