Showing papers in "Analytical Biochemistry in 1976"

Abstract: A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.

Abstract: A method for measurement of both oxidized (GSSG) and reduced (GSH) glutathione has been developed, with use of o-phthalaldehyde (OPT) as a fluorescent reagent. The method takes advantage of the reaction of GSH with OPT at pH 8 and of GSSG with OPT at pH 12; GSH can be complexed to N-ethylmaleimide to prevent interference of GSH with measurement of GSSG. The method gave “recoveries” of 91 to 110% for both GSH and GSSG and was quite specific for glutathione; and none of the manipulations appeared to influence the amount of glutathione present in the tissue. Results for GSH levels agreed well with earlier reports but levels of GSSG estimated here were higher than earlier reported values. The reasons for the apparently higher levels of GSSG are discussed.

Abstract: The Lowry protein assay is a sensitive but highly nonspecific procedure. The standard Lowry protein assay has been modified so that protein can be assayed in the presence of interfering chemicals. The method is based on the observation that in the presence of 125 μg/ml of Na-deoxycholate, bovine serum albumin (5—50 μg) is reproducibly precipitated (90–104%) by 6% trichloroacetic acid. Interference by sucrose, glycerol, Tris—HCl, Tricine, and EDTA can be eliminated. Protein samples containing carrier ampholytes can also be assayed provided their NaCl concentration is adjusted to 1 m .

Abstract: A method for agarose gel electrophoresis under denaturing conditions, with methylmercuric hydroxide as the denaturing agent, is described. Methylmercuric hydroxide is a strong and reversible denaturing agent. It appears that complete denaturation of any base-paired secondary structural feature of a nucleic aicd can be achieved at practical concentrations of CH3HgOH. The method has been tested by showing that singly nicked circular duplex PM2 DNA is dissociated into more rapidly migrating linear and circular single-strand forms by a CH3HgOH concentration within the 2.5–5.0 m m range. The mobilities of single-strand RNA molecules are decreased in the presence of sufficient CH3HgOH because their secondary structure is disrupted. For HeLa 28S rRNA, the melting transition occurs mainly in 2–3 m m range of CH3HgOH. For a series of RNA's, at 5 m m CH3HgOH, corresponding to complete denaturation, there is a linear dependence of log (molecular weight) on electrophoretic mobility and of log (mobility) on agarose concentration, just as in other gel electrophoresis systems.

Abstract: The use of 3,3′,5,5′-tetramethylbenzidine-H2O2 as a stain for the peroxidase activity of cytochrome P-450 (or cytochrome P-450 in sodium dodecyl sulfate polyacrylamide gels is described in this report. This reagent can be used to detect very low levels of heme-associated peroxidase activity. The blue-stained bands on polyacrylamide gels are distinet, and the color is stable. The stained gels can be photographed or scanned at 690 nm because the gel background remains clear. The stain is easily removed from the gels to permit subsequent protein staining. Staining first for peroxidase activity has no effect on the subsequent protein staining profile. The peroxidase activity of cytochrome P-450 (or cytochrome P-420) in immunoprecipitates in Ouchterlony double diffusion plates can also be detected using this reagent.

Abstract: A method utilizing precipitation on filter paper has been used to isolate glycogen from homogenates of liver or isolated hepatocytes. The procedure requires very small amounts of tissue or cell suspension and is rapid and highly reproducible. Its efficiency and specificity make it very suitable for many studies involving radioactive tracer incorporation into glycogen.

Abstract: A simple method is described in detail for the efficient isolation of high molecular weight covalently closed circular DNA (ccc-DNA) from Agrobacterium . Although this method was developed for isolating ccc-DNA of molecular weights greater than 10 8 daltons in Agrobacterium , the technique also proves to be useful in isolating ccc-DNA of varying sizes from a variety of other bacteria. The technique involves the shearing and alkali denaturation of the chromosomal DNA, followed by the preferential removal of the single-stranded DNA by phenol extraction. The DNA which remains is largely ccc-DNA. The DNA is then concentrated, and the ccc-DNA is separated from the chromosomal DNA by centrifugation in a cesium chloride-ethidium bromide density gradient. By this technique, ccc-DNA of varying sizes has also been isolated from species of Escherichia, Rhizobium, Citrobacter , and Lactobacillus .

Abstract: The inner and outer membranes of gram-negative bacteria are usually obtained from spheroplasts. The lysozyme treatments normally used for converting cells to spheroplasts were originally developed with exponential phase cells and have proven to be ineffective with cells grown under other conditions. A procedure has therefore been developed which renders variously grown cells completely susceptible to lysozyme. This procedure has been tested on various strains of Escherichia coli at all stages of growth in minimal medium, from the early exponential to the late stationary phase. It has been tested on stationary phase cultures which ceased to grow because of limiting aeration, limiting carbon source, limiting amino acids, and limiting nicotinic acid. Very efficient conversion of cells to spheroplasts was observed in all cases. The resulting spheroplasts are an excellent source for subsequent membrane separations.

Abstract: Methods for measuring total glutathione are described. These are based on the ability of glutathione and glutathione reductase (EC 1.6.4.2.) to catalyze the oxidation of NADPH by Ellman's reagent. Except for highest glutathione levels, NADP + rather than the reduced Ellman compound is measured. For intermediate sensitivity (2 × 10 −12 mol) NADP + is measured stoichiometrically by conversion to NADPH and determination of fluorescence. For smaller amounts (10 −14 mol) the NADP + generated is amplified by enzymatic cycling. These procedures have been tested extensively on kidney and are probably applicable to tissues in general.

Abstract: Cytochrome P -450 in whole liver homogenates, which contain an appreciable amount of hemoglobin, is detected by dithionite-difference spectroscopy of CO-bubbled homogenates. The molar extinction difference of cytocrhome P -450 by this method was determined to be 104 m m −1 cm −1 by comparative observations of the absorbance change in the dithionite- and CO-difference spectra of the membrane-bound hemoprotein. The content of cytochrome P -450 in normal rat liver was estimated to be 50 nmol/g wet weight of liver, and increased significantly after pretreatment of the animals with either phenobarbital or 3-methylcholanthrene.

Abstract: Nonspecific types of binding occur when oligo(dT)-cellulose is used to analyze or prepare poly(A)RNA. First, nonpolyadenylated nucleic acids bind and are eluted under conditions used to elute poly(A)RNA. Second, “tight” nonspecific binding occurs in which poly(A)RNA fails to elute under conditions which dissociate A-T bonds. Hydrolysis is required to remove tightly bound RNA. Oligo(dT)-cellulose has a low capacity for both these types of binding, and can be readily preempted with a heterologous RNA e.g., bacterial. Third, indirect nonspecific binding can also occur. rRNA aggregates with poly(A)RNA and thus can bind indirectly to oligo(dT)-cellulose. After these aggregates are disrupted by treatment with DMSO and heat, poly(A)mRNA free of rRNA can be isolated. Efficient recovery of poly(A)hnRNA from total nuclear RNA is accomplished using oligo(dT)-cellulose if the RNA is first subjected to conditions which disrupt aggregates and reduce secondary structure. Ninety-five to ninety-eight per cent of the purified poly(A)hnRNA and poly(A)mRNA rebinds to oligo( d T)-cellulose.

Abstract: Incubation of up to 8 μg of DNA with 30 mg DABA at 60°C for 30 min are the optimal conditions for development of fluorescence with DNA. At lower levels of DABA the time of incubation became crucial, and linearity at low DNA concentrations was lost. Application of the method to the estimation of DNA extracted with either 1 n KOH or 1 n PCA from monolayer cell cultures showed that fluorescence could still be developed satisfactorily in the presence of the alkali or acid without neutralizing or drying the sample, thus saving considerable time with no loss of accuracy or reproducibility of the assay, but with some loss of sensitivity.

Abstract: We have synthesized several new chromogenic substrates, p-nitroanilides of the dipeptides, Gly-Pro, Ala-Pro, Lys-Pro, Arg-Pro, Glu-Pro, and Asp-Pro, for X-prolyl dipeptidyl-aminopeptidase. These have permitted the development of a simple assay of the enzyme in which p-nitroaniline liberated directly or after the Bratton-Marshall reaction is measured spectrophotometrically. The enzyme activity was measured in human serum or in homogeneous enzyme purified from human submaxillary gland. The homogeneous enzyme hydrolyzed each substrate to produce X-Pro and p-nitroaniline. The optimum pH was at 8.7, except with Arg-Pro p-nitroanilide (8.0). Serum enzyme hydrolyzed Gly-Pro p-nitroanilide to p-nitroaniline and Gly-Pro, which was further hydrolyzed to Gly and Pro by an imidodipeptidase in serum. Gly-Pro β-naphthylamide or Gly-Pro-Leu was a competitive inhibitor with each X-Pro p-nitroanilide as substrate. Gly-Pro p-nitroanilide had the highest activity among the substrates at pH 8.7, followed by p-nitroanilides of Ala, Lys, Arg, Glu, and Asp in a decreasing order of activity.

Abstract: The time course of the bioluminescence obtained with a partially purified firefly luciferase preparation has been studied. At ATP levels less than 10−6m the light emission could be maintained essentially constant for several minutes, if the luciferase was not subjected to product inhibition or other inactivating processes. This could be achieved by performing the reaction at appropriate pH and concentration of luciferin and luciferase. Under these conditions continuous measurement of light emission may be used for nondestructive monitoring of ATP-converting reactions, since the emission will be proportional to the ATP concentration in each instant. The continuous monitoring of ATP concentration by firefly luciferase was used for kinetic determination of enzymes and metabolites and for endpoint analysis of metabolites. It was found to be extremely sensitive and convenlent for routine applications.

Abstract: The hydrolytic rate of p-nitrophenylphosphorylcholine by phospholipase C is highly accelerated in a medium containing sorbital. Glycerol is also effective in increasing the hydrolytic rate but does not increase it as much as sorbitol. We propose a convenient spectrophotometric assay method for phospholipase C using p-nitrophenylphosphorylcholine together with sorbitol (60%, w/w). This method was found very useful for studying the metal and detergent requirement of phospholipase C. The results obtained using this method suggest that phospholipase C of Clostridium perfringens is a metalloenzyme possibly requiring Zn2+ and that no type of detergent has any activating effect on the enzyme itself.

Abstract: After oxidation of indole-3-acetic acid with ceric nitrate in the presence of hydroxylamine, a stable colored complex extractable to isoamyl alcohol is formed. This reaction is useful for the colorimetric estimation of indole-3-acetic acid in biological mixtures. The colored complex (whose chromophore probably has similar structure as the product of the well known Salkowski's reaction) is relatively stable in light. The specificity of the method is discussed.

Abstract: A rapid micro-test using 2,4,6-trinitrobenzenesulphonic acid has been developed to detect incomplete coupling reactions in solid phase peptide synthesis. This new test will detect 3 nmol of free amino groups per milligram of resin.

Abstract: A new type of radioimmunoassay of cyclic AMP is described; it consists in taking into account the remarkable affinity of anticyclic AMP antibodies for 2′- O -succinyl derivatives of cyclic AMP, 100-fold higher than that for cyclic AMP itself. The samples to assay are submitted to a simple and rapid treatment which converts cyclic AMP into 2′- O -succinyl cyclic AMP with a 100% yield. Then, the radioimmunoassay is performed by equilibrium dialysis. The sensitivity is 100-fold increased, as compared to that of the usual radioimmunoassay. The specificity and the reproducibility are also improved. 10 −15 Mole cyclic AMP is routinely assayed, the minimum detectable being under 10 −16 mole.

Abstract: A new method that involves the use of ion-exchange resins to study ion transport in biological systems is presented. The transport of organic and inorganic ions was studied in a variety of biological systems including intact cells, sub-cellular particles (Such as mitochondria and plasma membrane vesicles), and reconstituted membrane vesicles. A comparison with such conventional methods as centrifugation and washing, gel filtration, and membrane filtration suggests that the method has certain advantages with respect to reproducibility, reliability, and convenience.

Abstract: The enzyme glutathione (GSH) peroxidase can be used to measure hydroperoxides quantitatively, easily, and specifically. A timed reaction of GSH peroxidase, coupled with the oxidation of NADPH by GSH reductase, allows a direct spectrophotometric measurement of hydroperoxide. Addition of catalase prior to the addition of GSH peroxidase permits the distinction between hydrogen peroxide and organic hydroperoxides. The solvents that can be used with the assay include methanol, ethanol, water, and aqueous solutions of detergents such as Brij 35, Triton X-100, and cetyl trimethyl ammonium bromide. The utility of the method is demonstrated by the measurement of hydrogen peroxide and organic hydroperoxides formed upon ozonolysis of an unsaturated fatty acid.

Abstract: A nondegradation method was developed for the isolation of elastin from tissues of 10 different animal species. Marked differences, involving primarily the neutral and aromatic amino acids, occurred in the amino acid composition of elastin of different species. The amino acid composition of elastins from a given species was constant for all the tissues studied.

Abstract: A sensitive method is described for the detection of glycoproteins after polyacrylamide gel electrophoresis. Dansyl hydrazine is condensed with aldehydic functions generated by periodate oxidation of protein bound carbohydrate. The resulting hydrazones are reduced to the stable hydrazine derivatives with sodium borohydride. After destaining, the fluorescent-labeled glycoproteins are visualized with a long-range uv light source. The dansyl hydrazine staining technique was shown to detect less than 40 ng of carbohydrate bound to Bandeiraea simplicifolia lectin. The principle methods for the detection of glycoproteins in polyacrylamide gels are the periodic acid-Schiff stain (PAS) (1,2,4) and the Alcian blue stain (3). Both of these procedures involve the fixation of the glycoproteins in the gel followed by oxidation of sugar residues with periodic acid generating aldehydic functions, reduction of the excess periodate with sodium metabisulfite and staining with either basic fuchsin or Alcian blue. The minimum amount of protein-bound carbohydrate which can be detected with these methods is in the range of 2-3 pg (4). The fluorescent reagent I-dimethylaminonaphthalene-S-sulfonylhydrazine (dansyl hydrazine) has been used for the detection and quantitation of carbonyl containing compounds such as keto steroids (5). We report here the use of this reagent as a sensitive fluorescent stain for the visualization of glycoproteins following polyacrylamide gel electrophoresis. The glycoproteins are subjected to periodate oxidation and the resulting adehydes condensed with dansyl hydrazine to form hydrazones. The latter are reduced to stable hydrazine derivatives with NaBH,.

Abstract: The intensity of staining of protein zones in polyacrylamide gels by Coomassie brilliant blue G250 in perchloric acid solution was increased by a factor of 3 when a wash of 5% acetic acid followed staining. Concentrations as low as 5 ng of human serum albumin could be detected in the gels.

Abstract: A new sensitive assay for amidase activity of chymotrypsin has been developed using 7-glutarylphenylalaninamido-4-methylcoumarin as substrate. Release of 7-amino-4-methylcoumarin was determined fluorometrically. As little as 0.5 μg/ml of chymotrypsin could be detected; at pH 8.0, K m = 0.7 m m . The substrate was not hydrolyzed by either trypsin or elastase and was capable of measuring chymotrypsin-like activity in tissue extracts. Hydrolysis of the substrate by chymotrypsin was blocked by specific inhibitors of the enzyme.

Abstract: Fluorescence of the thiobarbituric acid reaction product of periodate-cleaved sialic acid is readily detected by excitation at 550 nm and analysis of emitted light at 570 nm. A fluorometric microassay is described which is 500-fold more sensitive than the usual spectrophotometric procedures and readily detects 10 ng (32 pmol) of sialic acid. Contamination by 2-deoxyribose from cellular material is easily detected by shifts of excitation maxima below 550 nm.

Abstract: Collagen types I, II and III can be purified in their native state from heterogeneous collagen solutions by fractional precipitation at neutral pH using ammonium sulfate, sodium chloride and ethanol as precipitants. This method of collagen separation is useful as a preparative procedure and should also serve as an analytical tool for identification of unknown radioactively labeled collagens.

Abstract: A linear relationship was established between the log of the molecular weight of a protein incubated with 1% sodium dodecyl sulfate and the log of the polyacrylamide concentration reached by this protein after electrophoresis across a 3 to 30% polyacrylamide gradient gel. This relationship can be used to determine protein molecular weights from 20,000 to 1,000,000 with good accuracy.