Mayo Uziel

Rapid ion-exchange chromatographic microanalysis of ultraviolet-absorbing materials and its application to nucleosides☆

The major nucleosides obtained by hydrolysis of RNA are quantitatively assayed in less than 1 hr at the level of 1 or more nmoles by cation-exchange chromatography and a sensitive, continuous ultraviolet-absorbance monitoring device. A uniform cation-exchanger bead preparation (17.5 ± 2 μm diameter) permits operation at low (6–16 psi) pressures and rapid flow rates (1 ml/cm2 min) without loss of resolution. Analytical sensitivity is enhanced by improved flow cell and electronic circuit designs. A multi wavelength monitoring unit incorporated into the monitoring system aids in assaying substances of differing ultraviolet-absorption characteristics. Minor or altered nucleosides (20) and purine and pyrimidine bases (8) are separable, with few exceptions, by means of the single solvent chosen for the elution. The parameters determining the effectiveness of the separations have been explored and are expressed in terms permitting design modifications to improve the separation of refractory pairs or shorten the analysis time or both.

Surface-enhanced Raman analysis of benzo[A]pyrene-DNA adducts on silver-coated cellulose substrates

Benzo[a]pyrene-7,8-diol-9,10 epoxide (BPDE)-DNA adducts have been analyzed by surface-enhanced Raman scattering (SERS) spectrometry. BPDE is the ultimate carcinogenic metabolite of benzo[a]pyrene. The SERS measurements were performed with the use of a silver-coated cellulose material as the SERS-active substrate. The acid hydrolyzed products of BPDE-DNA adducts, DNA, and the BP-tetrols were also analyzed by SERS. The results indicate that SERS has great promise as a tool for detecting BPDE-DNA adducts in biological samples.

Preparation and properties of active, insoluble alkaline phosphatase

Abstract 1. One of the problems encountered in the sequential chemical degradation of nucleic acids is asynchrony arising from contamination by alkaline phosphatase during the oxidative elimination step. The residual enzyme activity persists because of its incomplete removal from the RNA during the preceding dephosphorylation step. If the enzyme can be converted into an insoluble active form, this problem can be avoided. 2. The coupling of alkaline phosphatase to several water-insoluble polymers has been studied. The polymers tested as support materials include: ethylene-maleic anhydride copolymers; polymethylvinyl-maleic acid anhydride copolymer; carboxymethylcellulose azide; linear and cross-linked polymers of methacrylic acid and methacrylic acid fluorodinitroanilide. 3. Preparations of the enzyme attached to all of the polymers except carboxymethylcellulose azide were fully active toward the synthetic substrate, p -nitrophenyl phosphate, and the four common nucleoside monophosphates (including 2′, 3′, and 5′ nucleotides). The activity toward nucleic acids is dependent upon the conditions of coupling.

Iodine modification of E. coli tRNAPhe: Reversible modification of 2-methylthio-N6-isopentenyladenosine and lack of disulfide formation

Abstract 1. The reaction products of potassium triiodide with E. coli tRNAPhe, tRNAVal, and tRNATyr indicate that, under given conditions, both 2-methylthio-N6-isopentenyladenosine and 4-thiouridine may be reversibly modified by thiosulfate. In contrast to reports by others, the tRNAs treated with triiodide and thiosulfate are active as amino acid acceptors. The modified tRNAPhe containing 2-methylthio-N6-isopentenyladenosine does not function in polyphenylalanine synthesis, but after thiosulfate treatment it becomes active. 2. Although tRNATyr forms a disulfide, neither tRNAPhe nor tRNAVal shows evidence of disulfide formation. The absence of disulfide formation in tRNAPhe and tRNAVal is indicated by the failure of the iodinated tRNAs to form 4-thiouridine sulfenic acid upon treatment with alkali, by the absence of disulfide upon direct analysis of the product after hydrolysis, and by no change in electrophoretic mobility upon gel electrophoresis, indicating the absence of intermolecular cross-links. 3. The 4-thiouridine reacts at least 10 times faster than the 2-methylthio-N6-isopentenyladenosine, so the two modifications are essentially independent and the thiouridine may be selectively modified. During the course of the slow reaction, 1.7 atoms of iodine are incorporated when about 70 % of the 2-methylthio-N6-isopentenyladenosine is modified. We believe that the modified adenosine is the site of incorporation even though three new components appear in chromatographic analysis, two of which disappear upon regeneration with thiosulfate, and incorporated 131I is not coincident with these new peaks.

Differences in the behavior of the purine and pyrimidine bases on dextran columns at different pH values

Abstract When the carboxyl groups of a highly cross-linked dextran gel are converted to their acid form, the gel can act as a cation exchanger towards purines and pyrimidines containing basic groups, and as a result the distribution coefficients of such compounds are markedly increased over those values obtained on neutral dextran. At alkaline pHs these same carboxyl groups interact in an opposite manner with the acidic groups of purines and pyrimidines to lower their distribution coefficients. Choosing a proper balance between the charge on the matrix of the gel, the charges of the compounds themselves, and the adsorptive effect of the compounds for the ether groupings of the dextran gel makes possible the analytical separation of purines and pyrimidines at acidic or alkaline pHs. Desalting of the purine and pyrimidine bases can be accomplished on dextran columns at neutral pH, since many of the common salts such as chlorides, acetates, formates, phosphates, and iodides precede the bases as a sharp and distinct band.

Measurement of DNA adducts using surface-enhanced Raman spectroscopy

Hazardous pollutants emitted from energy-related technologies, chemical industries, or waste materials are of increasing public concern because of their potential adverse health effects. Many pollutants have chemical groups of toxicological importance that can be characterized and detected by Raman spectroscopy. Raman spectroscopy, however, has not been widely used in trace organic detection, even though the information contained in a Raman spectrum is valuable for chemical identification. One limitation of conventional Raman spectroscopy is its low sensitivity, which often necessitates the use of powerful and costly laser sources for sample excitation. Raman spectroscopists have recently been able to analyze dilute biological samples as a result of enhancements in the Raman scattering cross section by factors up to 10(10) when a compound is adsorbed on or near a special electron-conducting surface. These spectacular enhancement factors of the normally weak Raman scattering process help overcome the low sensitivity of Raman spectroscopy through a combination of electromagnetic and chemical interactions between the analyte molecule and the surface. The technique associated with this phenomenon is known as surface-enhanced Raman scattering spectroscopy (SERS). The special conductive surface responsible for the scattering enhancement is referred to as a SERS substrate. For the past few years we have developed the SERS technique, using practical SERS-active substrate materials based on silver-coated microspheres deposited on glass. A wide variety of biomarkers have been investigated, including benzo[a]pyrene, dibenz[a,h]anthracene epoxides, 1,N9-ethenoadenine, 3,N4-ethenocytosine, and other substances. These biomarkers were measured at nanogram and subnanogram levels. The experimental results are of great analytical interest, since these chemicals are difficult to detect by other techniques, such as luminescence spectroscopy, because of the weak luminescence quantum yields of these DNA adducts. In this paper the potential usefulness of the SERS technique for assessing environmental and health effects from human exposure to toxic pollutants is demonstrated.

Synchronous Fluorescence Measurement of BaP Metabolites in Human and Animal Urine

Abstract Injection of Benzo(a)pyrene into rats results in urinary excretion of fluorescent metabolites of BaP. The synchronous fluorescence (SF) technique can detect picogram quantities of fluorescent compounds such as 7,8,9,10-tetrahydro-tetrahydroxy-benzo(a)pyrene (BPT). BPT was used as a model compound to show that SF can detect 0.1μM of polynuclear aromatic compounds (PNA) or PNA metabolites in urine by simple dilution. The relatively high concentration limit is due to the need to dilute the urine 100 fold before the characteristic pyrenyl spectrum is observed. The upper limit of direct quantification of BPT without dilution of whole urine is 5μM because of urine quenching effects. A 100-fold dilution of urine, containing added BPT, restores normal fluorescence and SF spectra. The 0.1 μM detection limit is lower than the observed amounts of individual PNA in nonexposed, nonsmoking humans. This approach may provide a simple, noninvasive tool for monitoring human exposure to fluorescent toxic substances.

Stability of 4,4′-di(thiouridine) and 4-thiouridine in alkali☆

Abstract Cation exchange chromatographic analysis can distinguish between the alkaline hydrolysis products of 4-thiouridine and 4,4′-di(thiouridine). Although 4-thiouridine is the primary stable alkaline hydrolysis product from either of the above compounds, the disulfide [4,4′-di(thiouridine)] yields a second, unidentified compund. This latter substance is less stable at 80° than at 37°.

Aryl hydrocarbon hydroxylase tissue‐specific activities: Evidence for baseline levels in mammalian tissues

The tissue-specific activities (units per gram tissue) of arylhydrocarbon hydroxylase benzo[a]pyrene [AHH(BaP)] (EC 1.14.14.2) in human, mouse, rat, and hamster have been reviewed. Three categories of AHH activities are defined: baseline values from tissues that have been protected from adventitious exposures to AHH inducers; background levels from tissues where there have been no overt measures to protect against exposure; and induced levels resulting from overt exposure to chemical inducers. Evidence that the baseline category exists is derived from the observations that an upper limit of AHH tissue-specific activity of about 1.5 nmol/h . g tissue occurs in human placenta, human foreskin, lymphocyte, and epitheliod and fibroblastoid cell lines; mouse lung and liver; rat fetal liver, and noninducible rat cell lines from lung, liver, embryo kidney, and adrenals; and hamster kidney. The collected values for nonexposed tissues range from 0.02 nmol/h . g to values less than 1.5 nmol/h . g. The most consistent observation of this type was from human placental material from nonsmoking mothers. Animals raised under standard laboratory conditions without special dietary precautions show background AHH activities that range from 2 nmol/h . g to 200 nmol/h . g in portal of entry tissues such as liver, lung, and intestines. Almost all tissue samples showed induced AHH levels of up to 500 nmol/h . g when those tissues were overtly exposed to substances containing chemical inducers of AHH. Measurements of placental AHH from smoking mothers showed that more than 95% of those samples had AHH values exceeding 2.5 nmol/h . g. This natural bimodal distribution of AHH activities, across species and in different tissues, of baseline values of less than 1.5 nmol/h . g and background or induced AHH activities with values greater than 1.5 nmol/h . g, may provide a reference set of values for use in quantification of the role of AHH in the induction of disease.

Analysis of Pseudouridine by Fluorescence Spectrometry

Abstract Fluorescence spectral properties of pseudouridine, an important biological indicator, have been measured for the first time in this study. The fluorescence quantum yield is maximal in aqueous solution with pH values above 9 with spectral properties characteristic of 5 alkyl substituted uracil derivatives. Chromatographic analysis shows the fluorescence is associated only with the pseudouridine peak. The concentration dependence of fluorescence is linear from 4 to 45 micromolar at pH 11.5. The excitation maximum is about 295 nm and the emission is broad with a maximum at about 390 nm. Addition of pseudouridine to urine extracts gives a linear increase in fluorescence with increasing pseudouridine concentration.