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We previously developed a method to detect the cellular ability of nucleotide excision repair, which functions to remove UV-induced lesions in DNA, using a plasmid-type fluorescent probe. A drawback to the popular use of this method was that the oligonucleotide containing the (6-4) photoproduct, which was used as a primer in the plasmid preparation, must be synthesized chemically. In this study, we prepared the probe using a post-synthetically UV-irradiated oligonucleotide as the primer. Transfection of cells demonstrated that this probe detected the repair ability of the cells in the same manner as the original probe.

We showed before that long linear DNA molecules containing single-strand interruptions and undergoing pulsed-field gel electrophoresis (PFGE) tend to break into subfragments (electrophoretic nick instability). Here we show that circular chromosomal DNA with single-strand interruptions remains in the wells during PFGE. This means that the presence of nicks in immobile circular DNA is not enough to break this DNA during PFGE. In other words, under the conditions of our study, the artifactual conversion of nicks into double-strand breaks that we detect in linear DNA does not contribute to the overall level of chromosomal fragmentation, as measured by PFGE.

In this study a simple and effective procedure for the isolation of individual neutral glycosphingolipids (GSLs) by preparative thin-layer chromatography is described. The method is based on nondestructive visualization of neutral GSLs on silica gel precoated thin-layer chromatography plates with anionic lipophilic fluorochromes. After thin-layer chromatography, individual neutral GSLs were detected by spraying the plate with fluorochrome solution followed by exposure to ultraviolet light. GSL containing silica gel was scraped off and extracted with chloroform:methanol:water (30:60:8, by vol). Neutral GSLs were freed from contaminating anionic fluorochrome by DEAE–Sepharose anion-exchange chromatography. Finally, a stepwise chloroform:methanol gradient chromatography on a small silica gel K60 column was employed to remove non-GSL impurities. Of nine different anionic fluorochromes tested, 2-( N -methylanilino)-naphthalene-6-sulfonic acid was found to be the most suitable for preparative purposes. The method was proved with reference GSL mixtures containing glucosyl-, galactosyl-, lactosyl-, globotriaosyl-, and neolactotetraosylceramides, each substituted with C 24 - and C 16 -fatty acids, resulting in isolation of individual GSL fractions. The technique was applied for the purification of neutral GSLs from mouse kidney and human granulocytes carrying Lewis x -epitopes. In summary, the method described offers an easy to handle and successful preparative thin-layer chromatography strategy to obtain pure neutral GSLs in microgram and milligram quantities.

A method for detecting the activity of ribonuclease inhibitors (RIs) after nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing was developed. Both types of electrophoresis were performed using vertical slab polyacrylamide gels in the presence of dithiothreitol and in a sealed system. In each system, purified 50 kDa human RI was visualized as a single band by immunoblotting with a specific antibody. RI activity in the polyacrylamide gel slab was detected by sandwiching the gel slab between a cellulose acetate membrane moistened with a solution of bovine pancreatic ribonuclease A and a dried agarose film sheet containing substrate yeast RNA plus ethidium bromide and incubating at 37°C. The ribonuclease penetrated the polyacrylamide gel and digested the substrate RNA in the agarose film. However, if an RI was present in the gel, the enzyme was inactivated by complex formation. Fluorescent bands corresponding to RIs were observed on a dark background under ultraviolet light. This activity staining had a high sensitivity allowing detection of less than 0.6 units of mammalian RIs (corresponding to 5 ng of purified human RI) and produced a sharp band which compared favorably with that obtained on immunoblotting. These electrophoretic techniques appear useful for the investigation of RIs in heterogeneous biological samples.

The new nonionic glycosidic surfactant N-octanoyl-beta-D-glucosylamine (NOGA, molar mass 305.37 g) was synthesized through an easy and efficient two-step procedure. Specifically, beta-D-glucosylamine was obtained by the replacement of the anomeric hydroxyl of D-glucose by an amino group which was then selectively acylated. NOGA was finally purified by silica gel column chromatography and recrystallization. This compound is stable and soluble in water and usual buffers up to 80 mM at 4 degrees C and up to 0.2 M at 37 degrees C. NOGA solutions are also characterized by a low ultraviolet light absorbance above 250 nm (epsilon 280 approximately 1.5 M-1 cm-1). Due to its very high critical micelle concentration (CMC = 80 mM, as determined by spectrofluorimetry), this surfactant may easily be removed from samples by dialysis or, to a lesser extent, by adsorption onto hydrophobic beads. Furthermore, NOGA is colorimetrically titrable by the ninhydrin method and its weak interference in protein determination by the bicinchoninic acid method is easy to overcome. This surfactant exhibits a good solubilizing power toward membrane proteins, with a marked selectivity for spiralin, a bacterial surface antigen. Protein extraction started below the CMC, but was much more effective above this concentration threshold. NADH oxidase activity, ligand binding by the glycine betaine-binding protein, and antigenicity of more than 20 membrane or soluble proteins were not altered by NOGA. Thus, owing to its extraction efficacy and mildness toward protein structure and activity, NOGA should prove useful for membrane studies and offers the additional advantage of being easy to synthesize at low cost.

Nine salicyl phosphate esters with hydrophobic substituents (5-phenyl, 5-(2,4-difluorophenyl), 5- tert -octyl, 5-cumyl, 5-(4- tert -butylphenyl, 5-(1-adamantyl), 5-( n -dodecyl), 5-(1,1-diphenylethyl, and 5-trityl) were synthesized and found to be good substrates for calf intestinal alkaline phosphatase. The enzymatic hydrolysis produced the corresponding salicylates, which were strongly fluorescent when excited by ultraviolet light around 300 nm with maximum emission at 420–435 nm. The salicylates were less soluble and/or more adhesive than the nonfluorescent salicyl phosphate substrates, resulting in localization of fluorescence signal, which is a requirement for membrane-based assays. The salicyl phosphates bearing 8–14 carbon substituents were found to be suitable detection reagents for dot-blot DNA hybridization assays on nylon membrane using a biotinylated probe, allowing the detection of 125 pg of target pBR322 plasmid DNA using a simple apparatus consisting of a transilluminator, a camera, and a 455-nm cutoff optical filter.

The bacterial cloning vector, pGreen-S, was constructed by inserting the enhanced green fluorescent protein (EGFP) gene at the XbaI restriction site of pUC18 plasmid. When expressed in Escherichia coli DH5alpha produced colonies that were an absinthe green color under daylight and strongly fluorescent green under longwave ultraviolet light. The pGreen-S vector was used to select for directional insert based on the loss of green fluorescence in recombinant colonies that was caused by the absence of EGFP. The EGFP reporter system differs from the conventional complementation of lacZ, making screening recombinants simpler, less expensive, and more effective.

A flow injection analysis involving a photochemical reaction and fluorometric detection has been developed for the determination of urinary kynurenic acid. Kynurenic acid was found to fluoresce on irradiation with ultraviolet light at pH 7.2 in the presence of hydrogen peroxide. This method was applied to flow injection analysis using a new procedure involving a "bypass line" for the simultaneous determination of urinary kynurenic acid and background fluorescence. The calibration graph showed linearity over the range of 0.20 to 120 pmol. For pretreatment of urinary kynurenic acid, a PRE-SEP C18 cartridge was used. The mean recovery of kynurenic acid from urine was 94.5%. The content of urinary kynurenic acid was 13.0 +/- 2.68 mumol/day. There was good correlation (r = 0.9729) between values determined by flow injection analysis and high-performance liquid chromatography.

Mounting evidence indicates that S-nitrosylation of critical cysteine residues in a protein represents a common feature of protein function regulation and cell signaling. However, the progress in studying the exact role of S-nitrosylation has been hampered by a lack of rapid and accurate methods for the detection of these S-nitrosylated proteins and the exact modification sites. In this article, we report a fluorescence-based method in which the S-nitrosylated cysteines are converted into 7-amino-4-methylcoumarin-3-acetic acid (AMCA) fluorophore-labeled cysteines-termed the AMCA switch method. The labeled proteins are then analyzed by nonreducing SDS-PAGE, and the S-nitrosylated proteins can be readily detected as brilliant blue bands after the activation of ultraviolet light. Furthermore, the sites of modification can be determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after in-gel tryptic digestion of the fluorescent band, and the recognizable AMCA tag in the MS spectra ensures the accurate site identification of the nitrosocysteines. Therefore, our method offers some apparent advantages by direct visualization of on-gel image of S-nitrosylated proteins, shorter experiment time by skipping the anti-biotin immunoblotting step, and elimination of the potential interference of endogenous biotinylated proteins. Based on this method, we detected the S-nitrosylation and the modified site in bovine serum albumin and gankyrin after in vitro S-nitrosylation. Overall, our results indicate that the AMCA switch method is a fast and accurate method to identify the S-nitrosylated protein and the modification sites.

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.

We have developed two new fluorescent dyes, SYPRO Orange protein gel stain and SYPRO Red protein gel stain, to detect proteins in electrophoretic gels. Stained protein bands can be excited by ultraviolet light at approximately 300 nm, or at visible wavelengths, with excitation maxima of 472 nm for the Orange stain and 547 nm for the Red stain. Detection can be documented with sensitivity similar to that achieved with silver stain, using standard UV transilluminators and Polaroid 667 black and white film, CCD cameras, or commercially available laser scanners. Staining with these dyes is noncovalent and is accomplished using a one-step procedure. Protein gels do not require fixation steps prior to incubation with the dyes. Staining is complete 30 to 60 min following electrophoresis, with no destaining required. Staining can also be accomplished by including dye in the running buffer; in this case a brief one-step destaining procedure follows electrophoresis. The dyes appear to bind to the detergent coat surrounding proteins in sodium dodecyl sulfate (SDS) denaturing gels; thus, staining in such gels is not strongly selective for particular polypeptides. Fluorescent signals are relatively photostable, allowing multiple photographs of gels to be taken without significant signal reduction.

Submandibular gland homogenates from age-matched male and female rats were focused on pH 4-6.5 isoelectric focusing gels. At least 14 bands (A to N) showing amidolytic activity against three different oligopeptide-7-amino-4-trifluoromethylcoumarin (AFC) derivatives, immobilized on cellulose diacetate overlay membranes, were observed under ultraviolet light on focused gels. All the proteinases were optimally eluted, from excised gel pieces, into 20 mM ammonium bicarbonate buffer, pH 9.8, containing 0.1% Triton X-100; the protein content of the band eluants was measured after selective precipitation of protein from interfering substances. Differences were observed in the substrate specificities of the proteinases such that enzymes in bands A and K showed increased reactivity to ZVKKR-AFC, and those in bands H and J to ZR-AFC and DVLR-AFC, respectively. While the ability of aprotinin to inhibit the enzymes showed an inverse relationship to their isoelectric points, soybean trypsin inhibitor was most potent against bands B to I and least effective against bands K to N. Extracts from the glands of female rats contained the same mixture of proteinases as their male counterparts but the concentrations of all the proteinases apart from band K were reduced by approximately 25-50%. Where suitable substrates are available these methods have general applications for the rapid identification and characterization of other enzymes fractionated on IEF gels.

A simple, inexpensive, and sensitive assay for peptidase activity has been devised. The assay was performed in a microtiter plate and was based on fluorogenic peptide substrates, many of which are commercially available. 7-Amino-4-methyl coumarin the fluorescent product liberated during an incubation period of between 1 and 16 h, was detected by inspection of the plate under ultraviolet light of wavelength 356 nm. A fluorometer was not required. Using alpha-chymotrypsin as a model enzyme, with succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-methyl-coumaryl-7-amide as substrate, it was shown that as little as 4 fmol of enzyme could be detected. The method was non-quantitative and was particularly suited to location of enzyme activity in fractions during a purification procedure. The validity of the assay was demonstrated by detection of activity of a known enzyme, alpha-chymotrypsin, after its purification by size-exclusion high-performance liquid chromatography. The method was used to locate two forms of aminopeptidase activity, in fractions from size-exclusion chromatography of an extract from reproductive tissue of Helix aspersa, using L-leucine 4-methyl-coumaryl-7-amide as substrate.

Girard-T reagent (trimethylaminoacetohydrazide chloride) under mild acid or alkaline conditions, reacts with aldehydes and ketones containing α-dicarbonyl functional groups to produce addition compounds which strongly absorb ultraviolet light. The glyoxal adduct has a λ max of 295 nm and an E max of 2.73 × 10 4 at pH max of 325 nm and an E max of 1.88 × 10 4 at pH > 9. As little as 5 nmol of glyoxal can be measured accurately; absorbance is linear with concentrations up to at least 3500 nmol, and the reaction is complete within 10 min. A variation of the method is described which permits the detection of certain α-dicarbonyl compounds on paper. Another variation allows the measurement of glyoxal in a glyoxal-guanylic acid adduct, a previously described modified nucleotide.

We describe a simple and sensitive method for staining of the carbohydrate moiety of glycoproteins in polyacrylamide or agarose electrophoretic gels. Gels are incubated in a solution of fluorescein-labeled concanavalin A. Following destaining with a neutral buffer, glycoproteins exhibit fluorescence under long-range ultraviolet light. Thus, the glucose/mannose containing β- and γ-chains of human fibrinogen give fluorescent bands, whereas the carbohydrate-free α-chain does not react. Less than 100 ng of hexose bound to fibrinogen β- or γ-chains could be detected. The procedure is suitable for staining of other carbohydrate residues in glycoproteins, which can be recognised by specific agglutinins, as shown by binding of fluorescein-labeled lectins from Ricinus communis to galactose residues of fibrinogen.

A new method for determination of 32Pi-ATP and [14C]ADP-ATP exchange rates is described. It is based upon separation of nucleotides and Pi by thin-layer chromatography on commercial aluminum or plastic sheets precoated with silica gel. The method permits avoiding special procedures for stopping of the reaction and for preparation of aliquots for thin-layer chromatography separation. It also allows to separate all adenine nucleotides and Pi in one chromatography procedure, and to work with a double label. The volume of the reaction mixture was 50-100 microliters. Aliquots (2-6 microliters) of the reaction mixture were taken at various moments without stopping the reaction and were layered immediately on a heated silica gel sheets. The nucleotides and Pi were separated in a solvent system which consisted of dioxane, isopropanol, 25% ammonia, and water (4:2:3:4, v/v). The nucleotide spots were detected in ultraviolet light, cut out, and their radioactivity was measured with a liquid scintillation counter. The method for measurement of kinetics of exchange reactions catalyzed by reconstituted into liposomes H+-ATPase complexes from beef heart mitochondria and high plant chloroplasts, is described.

A sensitive staining method for protein blots on nitrocellulose is described. It is based on the coupling of a fluorochrome, dichlorotriazynylaminofluorescein, to protein which yields products colorless in visible light but colored when protein blots are illuminated with long-range ultraviolet light. The coupling of a fluorochrome does not affect the antigenic properties of proteins and the stained blots can be subsequently probed with antisera. Thus, the method allows for the unambiguous identification of antigenic proteins transferred to nitrocellulose from sodium dodecyl sulfate-polyacrylamide gels.

The ultraviolet-light absorption and fluorescence of Triton X-100 were virtually eliminated by hydrogenation to its reduced cyclohexyl analog, RTX-100. The critical micelle concentration of RTX-100 was 12% higher than that of Triton X-100. RTX-100 and Triton X-100 were quite similar in their abilities to extract proteins from human erythrocyte membranes.

A simple method is described for visualizing submicrogram amounts of the cyanogen bromide cleavage products of proteins on sodium dodecyl sulfate-polyacrylamide gels. The peptide fragment mixture is conjugated with the fluorogenic reagent 2-methoxy-2,4,-diphenyl-3-(2H)-furanone prior to electrophoresis. The fluorescent peptide bands are visible under ultraviolet light, thus avoiding the need for fixation and staining. The determination of the structural homology of two immunologically related proteins is presented to illustrate this methodology.

The synthesis of the nitrene precursor 3-azido-2,7-naphthalene disulfonate (ANDS) is described. Upon exposure to ultraviolet light, the non-fluorescent azide photodecomposes into a reactive nitrene intermediate which can react with neighboring molecules to form fluorescent products. The reaction can be conveniently monitored by the development of fluorescence. The purity of the azide was determined by ion-paired, reverse-phase high performance liquid chromatography. Extinction coefficients, emission spectra, as well as nanosecond fluorescent lifetimes, where applicable, have been determined for the starting material, light-activated azide, and protein-bound reaction products. The use of 3-azido-2,7-naphthalene disulfonate as a hydrophilic surface probe of proteins and membrane structure is discussed.

Biliproteins and bilipeptides subjected to discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of zinc acetate form a complex which fluoresces an orange color when viewed under ultraviolet light. The complex between the bilin chromophore and the zinc ion fluoresces at wavelengths which can be selectively visualized in gels by using a red filter. For the biliproteins phytochrome and C-phycocyanin the minimum detectable quantities are 100 and 50 ng, respectively. This is comparable to the sensitivity of Coomassie blue staining. The technique has been used for selective detection of phytochrome in plant extracts and to distinguish chromophore-bearing peptides from those not containing chromophore in proteolytic digests of phytochrome.

The synthesis and testing of a new type of nucleic acid-protein photocrosslinking reagent is described. The reagents are composed of a psoralen ligand for nucleic acid photoattachment, which is linked to an azidobenzoyl group, for protein photoattachment. The linker contains a disulfide bridge which can be opened by reduction with mercaptans. The reagents were tested in a chromatin system, where it was found that they induced cleavable crosslinks between the histones and the DNA upon irradiation with long-wavelength ultraviolet light (λ > 300 nm).

An activity stain for the detection of pyridine nucleotide-linked dehydrogenases in polyacrylamide gels is described. Following incubation of the gel with substrate and cofactor, bands are visualized under ultraviolet light, where reduced cofactors fluoresce and oxidized cofactors appear black. The methods described are useful for any NAD- or NADP-linked dehydrogenase; the enzymes can be assayed in either the oxidative or the reductive direction. Also described is a preparative polyacrylamide gel system using the activity stain, which can be used as a general purification method for dehydrogenases. The preparative gels are crosslinked with bisacrylylcystamine. These crosslinks can be broken by the addition of thiols after the bands of interest have been located and excised. The protein of interest is then separated from the solubilized acrylamide by adsorption to a suitable resin.

EDTA binds to DEAE-cellulose columns and salt-eluted fractions may contain as much as 20 times the original concentration of EDTA. If not corrected for, this high concentration of EDTA will interfere with three of the most frequently used protein assays. The errors encountered were reductions in apparent protein concentration of 50% as measured by the Lowry assay, 25% by the biuret determination, and as much as 90% by the spectrophotometric method. The mechanisms for these interferences appear to be chelation of copper by EDTA, in the case of the Lowry and biuret assays, and differential absorption of ultraviolet light by EDTA in the spectrophotometric case. Several possible methods for overcoming these interferences are discussed.

Carbonic anhydrase was located on polyacrylamide gels with carbon dioxide as the substrate and bromocresol purple to indicate hydrogen ion formation. The developed gels were fixed by freezing to −70°, and the enzyme bands were detected by their low-temperature fluorescence in ultraviolet light.

A rapid method for the identification and estimation of CF is described. After separation on paper chromatograms, CF gives a grayish blue fluorescence under ultraviolet light on reaction with acidified orcinol. The compound formed is eluted with sodium bicarbonate solution and the fluorescence is quantitatively measured in a Klett fluorometer. This method has been used to estimate enzymically formed CF in an in vitro system of chicken liver homogenate. The possibility of developing the fluorescence in solution has also been demonstrated. Even 100 mμg of CF is detectable visually on paper chromatograms. In solution, 0.5–5.0 μg of CF can be assayed with Klett fluorometer.

The fluorescamine test for the rapid detection of trace amounts of uncoupled products from solid phase peptide synthesis is reported. This novel procedure can detect much smaller amounts of incomplete coupling with greater simplicity than has previously been possible. Since the test is carried out under mild conditions certain side reactions are circumvented. The fluorophor-resins are easily viewed under long wave ultraviolet light, are stable at room temperature, and may be used for quantitative evaluation.

A rapid assay method for the glyoxalase enzyme system in crude cellular extracts has been developed. The disappearance of methylglyoxal is spectrophotometrically measured after preparing the methylglyoxal disemicarbazone adduct, which strongly absorbs ultraviolet light at 286 nm. The adduct is formed rapidly and quantitatively at room temperature when a large excess of semicarbazide is mixed with methylglyoxal at neutral pH. The assay has been developed with rat liver homogenates but may be applied to any other biological system.

Methods are described for the in situ detection of histidase and urocanase after electrophoresis on polyacrylamide gel. The histidase method and the first of the urocanase methods involve photography of the gel in ultraviolet light after incubation with substrate. The second method for urocanase relies upon the reduction of 2,6-dichlorophenolindophenol by imidazolone propionate, the product of the urocanase reaction. The position of the enzyme is thus shown by a sharply defined transparent band on a blue background.

Radioactively labelled vitamin D 3 -4-C 14 was used as a tracer to study the in vitro degradation of vitamin D. Effects were noted of various environmental conditions such as temperature, dispersion into water, exposure to air, oxygen, and ultraviolet light, with chromatography on alumina and Fluoropak 80 being employed to evaluate destruction. Procedures which avoided potentially damaging environments are described for saponifying tissues and extracting them of vitamin D and possible metabolites. They consist basically of refluxing with KOH in methanol, extraction by organic solvents in the presence of adequate concentrations of alcohol, and partition prior to chromatography or radioactivity assay.

We describe a method for the direct visualization of nucleic acids on nylon membranes. Nylon is weakly fluorescent under short wave ultraviolet light allowing membrane-bound nucleic acids to be detected with a sensitivity of 10 ng. This procedure involves no staining or destaining of the gels prior to transfer, does not require duplicate sample lanes or blots, and does not interfere with transfer of the nucleic acid to the membrane or subsequent hybridization.

As described in this article, visualization and/or photography under uv light of 4-chloro-1-naphthol-developed, peroxidase-marked immunoblots allows an increase in sensitivity of more than 100 times over the apparent staining results observable under normal visible white light. This increase in sensitivity can be obtained with the minimal additional requirement of an uv lamp, with the actual chloronaphthol staining procedure remaining unaltered and thereby allowing the monitoring of specific reactions with much smaller quantities of antigen or antibodies. Substantial shortening of the procedure is another advantage, making it possible to complete in 20 min or even less a procedure usually requiring 3 to 6 h. The phenomenon depends on the uv absorption and the fluorescence quenching properties of the products of the peroxidase reaction. The absorption spectra of the membranes with or without peroxidase products indicate that an intermediate in the peroxidase reaction is responsible for the absorption under uv light. This intermediate accumulates under conditions where the final product absorbing in the visible light has not begun to be produced, thus explaining the large increase in sensitivity. The behaviors of three types of membranes, nitrocellulose, nylon, and Immobilon (PVDF), are compared. Due to its lower uv absorption, PVDF gives by far the best results, followed by nitrocellulose.

Trifluoroacetic acid (0.1% w v ) is an excellent solvent for polypeptides, is volatile, and has a low absorbance of ultraviolet light of low wavelength. Polypeptides subjected to chromatography on columns of TSK-G2000-SW or TSK-G3000-SW in this solvent were eluted as sharp peaks. Retention volume was dependent not only on molecular weight but also on the number of formal charges per molecule. For polypeptides with a similar molecular weight, that with the highest proportion of basic amino acid residues was eluted earliest. For the TSK-G2000-SW column, the degree of deviation from a linear relationship between elution volume and logarithm of molecular weight was directly proportional to the molecular weight to the power 2 3 divided by the number of positive charges per molecule. Inclusion of 0.25 m sodium chloride in the solvent increased both the upper and lower limits of the molecular weight range over which separation occurred. The use of 0.1% trifluoroacetic acid as the mobile phase is thus particularly applicable to the separation of peptides of low molecular weight.

A new method is described for accurately positioning the light source of the ultraviolet absorption optical system of the Beckman Model E analytical ultracentrifuge. This method provides very accurate determination of both the height and radial position. A six-channel centerpiece is used to produce a three-line image on the film. Densitometer scans of exposures made at several positions of the source are used to determine the optimum position.

A new surfactant, 6- O -( N -heptylcarbamoyl)-methyl-α- d -glucopyranoside (HECAMEG, molar mass 335.38 g), was synthesized by a simple and low cost procedure from methyl-α- d -glucopyranoside. This surfactant is characterized by a high solubility in water (even at 0°C), ultraviolet light transparency in the region useful for protein detection, and a high critical micellar concentration (CMC = 19.5 m m ), permitting fast elimination by dialysis. Furthermore, the surfactant is colorimetrically titratable by the anthrone technique and its weak interference in protein titration by the Lowry et al. procedure and the bicinchoninic method is easy to overcome. Two membrane proteins (NADH oxidase and succinate dehydrogenase) and a soluble enzyme (lactoperoxidase) retained full activity in the presence of HECAMEG below or above its CMC. The partial inhibition of β-lactamase (soluble form) by HECAMEG above the CMC was probably only apparent and due to an interference of the surfactant with the substrate rather than a direct effect on the enzyme. HECAMEG was capable of extracting up to 75% of bacteriorhodopsin from the purple membrane of Halobacterium halobium in a nondenatured form as indicated by the spectral properties of the protein. It also solubilized spiralin from the Spiroplasma melliferum membrane with a great selectivity and efficiency, without detectable loss of antigenic properties. These data show that HECAMEG is a very mild surfactant, useful for membrane protein studies.

A rapid method of screening chromatographic fractions has been developed for enzymes which metabolize fluorogenic substrates. Samples of the eluted fractions are applied to cellulose acetate gels and then incubated with the specific fluorogenic substrate. Fractions which possess enzymatic activity are visible as fluorescent spots when the gels are examined under long-wave ultraviolet light.

A number of fluorometric assays of hydrogen peroxide-producing oxidases are based on the formation of highly fluorescent products from homovanillic acid or related compounds by horseradish peroxidase. We report the observation that under continuous uv illumination at the wavelengths used for excitation in these methods, brain or muscle homogenates produce fluorescence increases in the absence of any exogenous enzyme substrate; when uv light is excluded, such increases are negligible. Arachidonic and linolenic acids also produce this effect. For this reason, measurements of H2O2 based on this principle are valid only if this nonspecific effect has been excluded, and should preferably be carried out as end-point rather than continuous assays. It is believed that the effect of uv light on the reaction is due to formation of H2O2 and/or oxygen free radicals, and polyunsaturated lipids appear to be involved as intermediates. Thus, the homovanillic acid-horseradish peroxidase system may prove useful in investigations of the effect of uv on the production of oxygen free radicals and lipid peroxidation.

A gel-permeation column of TSK-G3000-PW that was equilibrated and developed with 36 or 45% acetonitrile in 0.1% trifluoroacetic acid fractionated mixtures of peptides with high resolving power. In addition, the elution volumes of 11 standard peptides and proteins were linearly related to the logarithms of their molecular weights in the acetonitrile-trifluoroacetic acid solvent at both low and high flow rates. Since the solvent is volatile and relatively transparent to short-wavelength ultraviolet light this high-performance gel-permeation system offers a rapid and highly sensitive method for the analysis, characterization, and purification of peptides and proteins from complex mixtures.

A technique to quantitate small volumes of dilute solutions of different-sized DNA fragments has been developed. The detection limit was 0.7 μg/ml and the technique could be used even in the presence of diffusable substances, including those such as sodium dodecyl sulfate which affect surface tension and also exhibit fluorescence when stained with ethidium bromide and excited by ultraviolet light. The DNA was mixed with low-melting-point agarose and pipetted into preformed wells in an agarose plate, where it solidified. After diffusion of small molecules, the amount of DNA was estimated by comparing ethidium bromide-mediated fluorescence of samples with that of standards.

Plant peroxidase isozymes utilize hydrogen peroxide to oxidize redox dyes. Benzidine, the substrate most commonly used for identification of peroxidase isozymes, is a potent carcinogen and has been banned from laboratory use. O -Dianisidine, the other common substrate for peroxidase isozymes, is structurally related to benzidine and is a possible carcinogen. A peroxidase zymogram stain has been developed which uses eugenol, a substrate which is safe to use in the laboratory. Peroxidase isozymes are recognized as bright blue fluorescent bands under ultraviolet light and develop within 1 min after staining. Eugenol may also be used in a quantitative fluorimetric assay of peroxidase activity.

The photochemical modification of 16 S rRNA by longwave ultraviolet light in the presence of hydroxymethyltrimethyl psoralen (HMT) produces uridine-psoralen adducts. Psoralen-modified 16 S rRNA is primed with a 3′-proximal restriction fragment and copied with reverse transcriptase. The average length of the cDNA transcripts decreases as the number of covalently bound psoralen adducts on the template RNA increases. Adducts stop reverse transcriptase and abbreviated cDNAs terminate at the positions of the lesions. The cDNA reverse transcripts of psoralen-reacted 16 S rRNA are electrophoresed in parallel with a dideoxy sequence (Sanger method) which uses the same restriction fragment as a primer. The psoralen-induced cDNA stops are found to be highly localized hot spots. In the 150-nucleotide sequence investigated at sequence resolution, two hot spots for psoralen reaction are found. In both cases the psoralen-induced stops are at UpU sequences near the bases of predicted hairpins.

Two versions of an approach to identify DNA-protein interactions at sites of DNA replication in HeLa cell nuclei are described. In this procedure, newly replicated DNA chains are first labeled and photosensitized in vitro by the incorporation of [alpha-32P] dCTP and bromodeoxyuridine triphosphate, respectively. Irradiation with ultraviolet light is then used to covalently crosslink the proteins that are adjacent to the photosensitized and isotopically labeled strands of newly replicated DNA. After the bulk of the DNA is digested with nucleases, the crosslinked proteins--marked by short covalently linked radioactive DNA tags--are fractionated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and detected by autoradiography. With this technology, certain proteins have been shown to associate selectively with newly replicated DNA. The method appears adaptable for application to a variety of problems involving DNA-protein association.

A simple method of determinations of three hexosamines is presented. The solutions of hexosamines heated in 1 n NaOH at 60°C for 60 min, absorb ultraviolet light with the maximum at 302 nm. A linear relationship exists between the concentration of hexosamine up to 100 μg and the absorbance at 302 nm. N-acetylated hexosamines, having been deacetylated, may be determined in this way. The characteristic absorption curve ranging from 225 to 360 nm with the maximum at 302 nm may be utilized as a qualitative criterion for identification of hexosamines and N-acetylated hexosamines after their deacetylation.

A method is described for the selective detection of keto sugars on paper chromatograms. After the chromatograms are developed, they are sprayed with an ethanol solution of dimedon and orthophosphoric acid, dried, and heated to 110°C for 5 min. When viewed under white light and ultraviolet light, spots containing keto sugar show a typical dark gray color and dark pink fluorescence, respectively, which are stable for several weeks. The minimum sensitivity of the solution is about 1 μg of ketose.

A method has been developed for the isolation of ultraviolet light induced pyrimidine dimers from enzymic hydrolyzates of DNA. The dimers are isolated in the form , thus making it possible to use 32P-labeled DNA for the study of ultraviolet light induced pyrimidine dimers.

A procedure is described for detecting particles containing nucleic acid following centrifugation of the particles in sucrose or potassium tartrate density-gradient columns. Quartz centrifuge tubes containing the gradient columns were photographed in ultraviolet light after centrifugation. The method has been used to identify 12 μg of tobacco mosaic virus and 2 μg of turnip yellow mosaic virus.