Improved estimation of DNA fragment lengths from Agarose gels

A simple, sensitive assay for prolylcarboxypeptidase (PCP) is described. It utilizes a radiolabeled substrate, benzyloxycarbonyl-l-prolyl-l-[³H]alanine, and the details of its synthesis are also reported here. The hydrolysis of the dipeptide substrate is linear with respect to time or protein concentration until 10% of the substrate has been cleaved. Kinetic analysis yielded a K_m of 4.7 mm. The assay can be used to measure PCP activity in small amounts of biological fluid, homogenized tissue or cultured cells. Measurements of PCP activity in various cultured human cells showed endothelial cells from umbilical veins to have the highest activity (1625 ± 151 nmol/mg/h) followed by endothelial cells from umbilical artery (1017 ± 46 nmol/mg/h), human foreskin fibroblasts (719 ± 39 nmol/mg/h), and pulmonary artery endothelial cells (352 nmol/mg/h).     

Direct hybridization of labeled DNA to DNA in agarose gels

A naringinase assay capable of distinguishing between the content of naringin, prunin, and naringenin present in the incubation mixture, is described. The amount of these compounds can be estimated by combining two spectrophotometric procedures. (a) Treatment with strong alkali to determine the amount of nargingenin as well as the sum of naringin and prunin. (b) Assay of the liberated aldohexoses with o-aminodiphenyl. From the data thus obtained, the amount of the remaining substrate, the amount of the intermediate as well as the product at any given time can be calculated.     

Visualization by chilling of protein bands in polyacrylamide gels containing 8 M urea: preparation and quantitation of foot-and-mouth disease virus capsid proteins

Protein bands become visible in polyacrylamide gels containing 8 m urea after chilling the gels in air for 5 to 10 min at ?70°C. Urea appears to crystallize preferentially as opaque bands in regions of the gel where protein reduces the amount of free water available as solvent for the urea molecules. Thus detected, the gel sections containing protein bands from foot-and-mouth disease virus can be immediately cut out, and their proteins obtained by electrophoretic elution or extraction procedures. Analysis of the proteins for purity and concentration is then carried out by electrophoresing measured aliquots on analytical gels, staining with Coomassie brilliant blue, scanning the gels for absorbance at 600 nm, and converting peak areas to micrograms of protein using Folin phenol standard curves determined for each purified capsid protein. The most basic capsid protein and its in virion proteolytic-cleavage products stain metachromatically.     

Two-dimensional separation of phosphoamino acids from nucleoside monophosphates

A comparative analysis of the migration of phosphoamino acids and nucleoside monophosphates in three different two-dimensional systems is presented. The three phosphoamino acids studied are phosphoserine, phosphothreonine, and phosphotyrosine, which are the residues most commonly occurring in proteins phosphorylated by protein kinases. Their migration properties have been compared to those of UMP, AMP, CMP, GMP, and TMP, which are the basic components of nucleic acids. Special attention has been paid to the behavior of UMP, which has previously been reported to often co-migrate with phosphoamino acids. Also, the migration of inorganic orthophosphate and ribose monophosphate, which are frequently present in samples derived from macromolecule hydrolysis, has been analyzed. The following separating systems have been used: double chromatography, electrophoresis followed by chromatography, and double electrophoresis. The latter is shown to have the best resolving power and to be the most convenient system.     

Fluorometric quantitation of single-stranded DNA: a method applicable to the technique of alkaline elution

The use of Hoechst dye 33258 for the fluorometric quantitation of single-stranded DNA was investigated for the purpose of developing a simple nonradiometric method of quantitating DNA in fractions collected during the analysis of DNA damage by the method of alkaline elution. The sensitivity of the assay allowed amounts of single-stranded DNA as small as 100 ng to be quantitated reliably. The requirement of a near-neutral pH necessitated that alkaline samples be buffered in order to perform DNA quantitation. However, that the addition of a predetermined volume of buffered dye solution to each sample is the only manipulation required prior to fluorescence measurement makes this procedure the simplest yet described for quantitating DNA collected during alkaline elution.     

Rapid electroelution of nucleic acids from agarose and acrylamide gels

The alkaline/filter DNA elution technique measures single-strand DNA breaks in mammalian cells based on the DNA molecular weight dependent retention of the macromolecule on 2-μm-pore-size filters. Described here is a modification of the technique which uses [³H]thymidine-labeled DNA of γ-irradiated cells as an internal reference. Thus, an increased precision is obtained in the assessment of this type of DNA damage at biologically significant radiation doses (i.e., where cell survival occurs). The measure of DNA damage is based on the actual initial DNA elution rate, i.e., arithmetic ratio of the elution of “test” DNA (i.e., ¹⁴C-labeled DNA) relative to the elution of “reference” DNA (i.e., ³H-labeled DNA). The repair of this damage on postirradiation incubation of the cells is detected as a decrease in the rate of “test” DNA cluted relative to “reference” DNA from unincubated cells. For Chinese hamster V79–171 cells irradiated with 5 Gy (500 rads), repair can be resolved into two first-order processes having rate constants (at 24°C) of ～0.190 and ～0.017 min^?1.     

A comparison of van't Hoff and calorimetric heats of binding to DNA using an ethidium selective electrode

A liquid membrane electrode selective for ethidium ion was used to measure free ethidium in mixtures with calf thymus DNA. Electrode response was unaffected by variation in ionic strength from 1 mm to 0.5 m, and was not degraded over the temperature range studied. DNA-ethidium binding isotherms obtained with the electrode at 17.4, 25.4, 30.1, and 40.6 °C were fitted to a single class of excluded sites model for $\text{?}$gn ranging from 0.01 to 0.16. van't Hoff analysis of these data yielded ΔH = ?8300 cal/mol ethidium bound (in 0.5 m KCl, 10 mm Tris buffer, pH 10, 1 mm EDTA). Direct calorimetric measurements of the heat of complex formation led to a value of ?7600 cal/mol at 25 °C in the same medium; the two results were not significantly different at the 95% confidence level. The agreement supports the validity of the ethidium selective electrode, and illustrates its utility in the study of ligand binding to nucleic acids and related materials.     

Activity staining of nucleolytic enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Use of aqueous isopropanol to remove detergent from gels

The sensitivity with which RNase and DNase activity can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) varies widely, depending upon the particular SDS preparation used for electrophoresis. (See also [10.], Anal. Biochem. 100, 357–363.) Sensitivity of detection is greatly increased by using buffered 25% isopropanol, rather than buffer alone, to wash detergent from gels after electrophoresis. Thus it is routinely possible to detect bovine pancreatic RNase A at the picogram level. Use of isopropanol improved activity staining of RNases with each of the 10 SDS preparations examined, including one containing 32% tetradecyl sulfate and 4% hexadecyl sulfate, and reduced the variability from preparation to preparation observed when buffer alone was used to remove SDS. Other water-organic cosolvent binary mixtures can be used but none shows advantages over aqueous isopropanol when sensitivity of detection as well as availability and cost of organic solvent are considered.     

Improvements of DNA sequencing gels

We describe three simple modifications of DNA sequencing gels which all result in improved oligonucleotide resolution as visualized by autoradiography. First, it was possible to reduce the thickness of the gel to 0.2 mm by using new gel molding techniques. Second, the gel could be dried without any distortions of its dimensions by prior binding of the gel to the surface of the glass plate. Third, a uniform high temperature was obtained in all parts of the gel during electrophoresis by replacing one of the glass plates with an inexpensive thermostating plate with circulating water. The use of this heating plate resulted in a straight band pattern all over the gel and also in the resolution of such bands which were not resolved in other electrophoresis systems.     

Characterization and quantitation of myosin heavy-chain mRNA from embryonic cardiac tissue

The messenger RNA (mRNA) coding for myosin heavy chain from the 16-day-old chick embryonic cardiac tissue was purified by a rapid isolation procedure and characterized. The mRNA can be translated with fidelity under optimally chosen conditions. The protein synthesized in response to the RNA was a polypeptide of 200,000 molecular weight, identical to the authentic myosin heavy chain from the homologous chick heart tissue. The purity of the mRNA was assessed by electrophoresis in denaturing gels, by immunoprecipitation of the translation product, and by analysis of the kinetics of hybridization with the complementary DNA (cDNA). The cDNA reassociated with myosin heavy-chain mRNA with kinetics characteristic of a pure mRNA. The sequence complexity data indicated that in the 16-day-old chick embryonic heart cells there is a single mRNA sequence coding for myosin heavy chain in contrast to two different mRNA sequences reportedly present in the skeletal muscle cells (M. Patrinou-Georgoulas and H. A. John, 1977, Cell12, 491).     

Affinity electrophoresis: New simple and general methods of preparation of affinity gels

Two simple and generally applicable methods of preparation of affinity gels for affinity electrophoresis in agarose and polyacrylamide gels are described. In the first method, amino ligands are coupled to periodate-oxidized agarose gel beads (Sepharose 4B), and homogeneous affinity gels are obtained after mixing the melted substituted beads with either melted agarose solution or with the polymerization mixture used for the preparation of polyacrylamide gels. This type of affinity gel was used for affinity electrophoresis of lectins (immobilized p-aminophenyl glycosides), ribonuclease (immobilized uridine 3′,5′-diphosphate 5′-p-aminophenyl ester), trypsin (immobilized p-aminobenzamidine), and double-stranded phage DNA fragments (immobilized acriflavine). Alternatively, heterogeneous affinity gels are prepared from the suspension of ligand-substituted agarose, dextran, or polyacrylamide gel beads in the polymerization solution normally used for preparation of polyacrylamide electrophoretic gels. This technique was used for affinity electrophoresis of lectins, ribonuclease, and trypsin on affinity gels containing appropriate ligands coupled to the gel beads “activated” by various methods. Applicability of affinity gels prepared by the two methods described above for affinity isoelectric focusing is demonstrated.     

Simultaneous extraction and assay of cyclic nucleotides, prostaglandins, and DNA from cat alveolar bone

A method for the simultaneous extraction of cAMP, cGMP, PGE₂, PGF_2α, and DNA from a small sample of mineralized bone and the subsequent assay of these substances is described. Various solvents were tested for efficiency of extraction for the fatty acids, and water or 40% ethanol was found to extract more than 90% of labeled prostaglandin. In order to avoid enzymatic degradation, the substances were extracted at ?5°C requiring a solvent which would not freeze during extraction. Frozen alveolar cat bone samples were homogenized in 40% ethanol in the presence of 5 mm EDTA to inhibit phosphodiesterase. Small aliquots of the homogenate were withdrawn for the spectrofluorophotometric assay of DNA. After centrifugation, the supernatant was extracted first with petroleum ether, in order to take out neutral lipids, followed by ethyl acetate partition. The ethyl acetate layer was dired with N₂ gas, reconstituted with assay buffer, and assayed for PGE₂ and PGF_2α. A portion of the aqueous fraction was used for cAMP binding assay, while the rest was column chromatographed to elute the cGMP for radioassay. On the basis of per microgram of DNA, values for each of the following in cat alveolar bone were: 0.346 ± 0.049 pmol for cAMP, 0.026 ± 0.001 pmol for cGMP, 5.52 ± 1.46 pg for PGE₂, and 1.00 ± 0.29 pg for PGF_2α. Values calculated after the dilution of the sample aliquots or addition of standards to cAMP, cGMP, or PGE₂ showed no significant difference (P < 0.05) to their respective values. Within the limits of the sensitivity for each of the assay systems, it is feasible to measure cAMP, cGMP, PGE₂, and PGF_2α in alveolar bone from the same sample.     

A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria

A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.     

Immunocapillarymigration--a new method for immunochemical quantitation.

A new simple and rapid method for immunochemical quantitation called immunocapillarymigration is described. It is based upon the attachment of antibodies to a porous insoluble support and the subsequent capillarymigration of the antigen-containing solution in the porous support. The migration of the antigen solute is specifically delayed in comparison to the migration of the solvent and other solutes in the process and the relative delay decreases with increasing antigen concentration. When applied to the quantitation of transferrin in human plasma, immunocapillarymigration gave results which agreed with those obtained by single radial immunodiffusion.     

Ultrathin-layer agar gels: a novel print technique for ultrathin-layer isoelectric focusing of enzymes

A novel, rapid, and simple print technique for ultrathin-layer isoelectric focusing of enzymes consisting of ultrathin-layer agar gels on polyester films is presented. Details for preparation of agar gels and the application for detection of commercial amylase, protease, pectinesterase, and lipase are given.     

Staining for protease activity on polyacrylamide gels

A high-performance liquid chromatographic procedure has been developed for the detailed analysis of amino acids and related compounds in 10-μl samples of perilymph from the guinea pig cochlea (inner ear). The procedure employs an Aminco amino acid analyzer and combines the use of a single chromatographic column, lithium citrate buffers for elution, a change of column temperature, and fluorometric detection of o-phthaldialdehyde/2-mercaptoethanol adducts of primary amines. Sensitivity is about 0.2 pmol referenced to leucine. Fifty-four primary amine components are detectable in perilymph collected in relative silence. Twentynine compounds have been identified, and six are putative amino acid neurotransmitters. The present method provides new information about the chemical composition of perilymph and is suitable for the analysis of physiological fluids available only in volumes of several microliters.     

Determination of the formal reduction potential, the electron stoichiometry, and the magnitude of the conformational change upon reduction for cytochrome c from potential-dependent fluorescence spectra

A long-optical-path electrochemical cell was used to determine the formal reduction potential (E0') and the electron stoichiometry (n) for 1 microM horse heart cytochrome c solutions from Nernst plots of the fluorescence spectrum of the tryptophan-59. Various concentration ratios of the oxidized to the reduced forms of the protein were generated by application of different potentials. The fluorescence spectrum was found to decrease with increasing concentrations of reduced cytochrome c. This decrease is interpreted in terms of a movement of the tryptophan toward the heme upon reduction, resulting in an increase in the rate of energy transfer to the heme. The magnitude of the conformational change is compared for cytochrome c in NaCl-H2O and NaBr-H2O solutions with the latter being larger.     

4-Methylumbelliferyl-glycosides as fluorescence probes of sugar-binding sites on lectin molecules: spectral properties and dependence of fluorescence on polarity and viscosity

The spectral properties of 4-methylumbelliferyl-glycosides (MeUmb-glycosides) were investigated in order to assess their usefulness as probes of the microenvironment of sugar binding sites on lectin molecules. It was shown that the abnormally high values for fluorescence polarization of free MeUmb-glycosides (from 0.07 to 0.251) were due neither to their molecular size nor to the blockade of their movement, but to the short lifetimes (less than 0.55 ns) of the excited state of these compounds. Working essentially with two MeUmb-monosaccharides and one MeUmb-disaccharide (MeUmb-alpha-D-galactopyranoside, MeUmb-beta-D-galactopyranoside, and MeUmb-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-beta-D- galactopyranoside) which were solubilized in various solvents, it was demonstrated that solvent polarity and viscosity definitely affected the fluorescence intensity of MeUmb-glycosides. A low-polarity medium reduced this intensity, and high viscosity enhanced it. The implications of these findings are discussed in relation to the variations in the fluorescence intensity of MeUmb-glycosides when these compounds were bound to lectins.     

Radioimmunolocalization of murine mammary tumor virus proteins in gels

Nycodenz is a new nonionic iodinated gradient medium which readily dissolves in water to give nontoxic, autoclavable solutions. This paper describes the use of diffusion techniques to prepare isotonic Nycodenz gradients with maximum densities up to 1.15 g/ml, which is sufficient to band most types of cells.     

Two-dimensional, 1H-n.m.r. study of peracetylated, reduced derivatives of three oligosaccharides isolated from human milk

The structures of the peracetylated derivatives of the following alditols obtained from oligosaccharides of human milk have been established by two-dimensional, J-resolved and J-correlated, ¹H-n.m.r. spectroscopy at 360 MHz: β- d-Galp-(1→3)-β- d-GlcpNAc-(1→3)-β- d-Galp-(1→4)- d-Glc-ol, α- l-Fucp-(1→2)-β- d-Galp-(1→3)-β- d-GlcpNAc-(1→3)-β- d-Galp-(1→4)- d-Glc-ol, and β- d-Galp-(1→3)-β- d-GlcpNAc-(1→3)-[β- d-Galp-(1→4)-β- d-GlcpNAc-(1→6)]-β- d-Galp-(1→4)- d-Glc-ol.