Improved oligonucleotide sequencing by alkaline phosphatase and exonuclease digestions with mass spectrometry

The combination of exonuclease digestion and mass spectrometry has been widely used for sequencing oligonucleotides. During an exonuclease digestion, rapid buildup in the concentration of nucleotides produces strong signal of nucleotide cluster ions in electrospray ionization-mass spectrometry, especially for oligonucleotides with greater than 25 bases. This leads to poor signal/noise ratio in the reconstructed molecular weight spectra of late digestion products due to artifact peaks from nucleotide cluster ions. Here we report a procedure that eliminates the effect of the cluster ions. In this method, alkaline phosphatase is added with snake venom phosphodiesterase to the oligonucleotide solution to convert the interfering nucleotides into noninterfering nucleosides, and the collision-induced dissociation spectrum of the dimeric oligonucleotide at the end of the digestion is obtained to determine the sequence of the last two bases at the 5'-terminus of the oligonucleotide. With this approach, the signal/noise ratio of the reconstructed molecular weight spectrum is greatly improved for relatively large oligonucleotides, and only a single digestion is needed for sequencing.     

Mapping nucleotide binding site of calcium ATPase with IR spectroscopy: effects of ATP gamma-phosphate binding

The changes in the IR spectra of the sarcoplasmic reticulum Ca2+-ATPase upon nucleotide binding are recorded in H2O at 1 degrees C in different buffers [imidazole, methylimidazole, 3-(N-morpholino)propanesulfonic acid, and phosphate] at different pH values (pH 6.5-7.8). The difference spectra of nucleotide binding are sensitive to the composition of the solvent. With methylimidazole at pH 7.5 providing the largest binding-induced signals, the effects of gamma-phosphate binding are investigated using ATP, ADP, and beta,gamma-iminoadenosine 5'-triphosphate. The gamma-phosphate contributes approximately 20% to the conformational change seen by IR spectroscopy and affects the beta-sheet structures. The IR experiments also reveal the known affinity difference between ADP and ATP.     

Determination of tryptophan, tyrosine, and phenylalanine by second derivative spectrophotometry

Second derivative spectrophotometry has been useful for the determination of aromatic amino acids. However, published methods produce erroneous results, because those methods measure second derivative values by the vertical distance between peak and trough which is subject to variation according to the aromatic amino acid composition of proteins. This paper presents a method of second derivative spectrophotometry which measures second derivative absorbance values by means of the vertical distance from baseline to the derivative curve at a wavelength specifically assigned to each aromatic amino acid, and makes corrections for the interference from other amino acids at the same wavelength. The Appendix describes a computational method for obtaining absolute values of second derivative absorbances directly from normal absorbance values without using the spectrophotometer's derivative mode, because most commercial instruments produce completely arbitrary second derivative values which make comparison of data obtained on two different instruments impossible.     

Magnetic circular dichroism studies on acid and alkaline forms of horseradish peroxidase.

The heme vicinities of the acid and alkaline forms of native (Fd(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III)protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentocoordinated heme complex. The MCD spectra of native horseradish peroxidase did not shown any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.     

Analysis of polysaccharide-poly(ethylene-co-acrylic acid) composites by fourier-transform infrared spectroscopy

A method, based on absorbances in FTIR spectra of the C---O singlebond stretch band of the polysaccharide and the C---H stretch band of poly(ethylene-co-acrylic acid) (EAA) at 2851 cm⁻¹, was developed for the determination of EAA in composites with either dextran or starch. Spectral subtraction of the polysaccharide component was necessary for quantitative determination of EAA. The accuracy of this analytical procedure is affected by the fact that absorbances of these two bands are not equally dependent on particle size of the sample in the KBr pellet; the absorbance ratio, as calculated from FTIR spectra therefore varies with sample preparation conditions. To ensure the necessary particle size uniformity during the preparation of KBr pellets, a method based on relative intensities of two bands in the C---O region of the spectrum was developed as an indicator of particle size in the pellet. This method was also used for monitoring sample size in the polysaccharide standard used for spectral subtraction, since particle size uniformity between sample and polysaccharide standard was also necessary for accurate determination of EAA.     

Leghemoglobin. Low temperature optical spectra of acid and alkaline forms of leghemoglobin(IV). Configuration of the heme.

Leghemoglobin(IV), the derivative of leghemoglobin at the formal oxidation state IV, when cooled to liquid nitrogen temperature exhibits radically different spectra at acid and alkaline pH. The acid and alkaline forms are freely interconvertible. The optical spectrum of the acid form is closely similar to optical spectra of the red higher oxidation states of horseradish and cytochrome c peroxidases, showing that the configuration of the heme iron is the same throughout this family of compounds. That configuration is believed to be Fe(IV) in a porphyrin environment. The optical spectrum of the alkaline form of leghemoglobin(IV) recalls that of alkaline low spin ferric leghemoglobin. Near infrared spectra of leghemoglobin(IV), myoglobin(IV), and the higher oxidation states of the peroxidases are featureless to 1300 nm, suggesting a common structural feature. The acid form of leghemoglobin(IV), seen in fluid buffer as a transient species at pH 5 or less, is conveniently generated by cooling a solution of the more stable alkaline form in borate buffer to liquid nitrogen temperature. At this temperature borate buffers become acid.     

A new approach to the study of nucleotide sequences in DNAs

The composition of the 3′terminal, 5′terminal and 5′penultimate nucleotides of the oligonucleotides released by spleen acid DNase and snail acid DNase from five `repetitive' DNAs (guinea pig, mouse and crab satellite DNAs, yeast mitochondrial DNA and poly (dAT:dAT)) and four `eukaryotic' DNAs (calf thymus, guinea pig and mouse liver DNAs, and yeast nuclear DNA) have been investigated and found to deviate in characteristic ways from those expected for bacterial DNAs having comparable base compositions. The deviation patterns obtained represent a novel way of characterizing and comparing different DNAs on the basis of the frequency of the nucleotide sequences they contain.     

Identification of the ligand-exchange process in the alkaline transition of horse heart cytochrome c.

Magnetic-circular-dichroism (m.c.d.) spectra over the wavelength range 300-2000 nm at room temperature and at 4.2K of horse heart cytochrome c are reported at a series of pH values between 7.8 and 11.0, encompassing the alkaline transition. The effect of glassing agents on the e.p.r. spectrum at various pH values is also reported. Comparison of these results with spectra obtained for the n-butylamine adduct of soybean leghaemoglobin support the hypothesis that lysine is the sixth ligand in the alkaline form of horse heart cytochrome c. The m.c.d. and e.p.r. spectra of horse heart cytochrome c in the presence of 1-methylimidazole have also been examined. These studies strongly suggest that histidine-18, the proximal ligand of the haem, is the ionizing group that triggers the alkaline transition. Low-temperature m.c.d. and e.p.r. spectra are also reported for Pseudomonas aeruginosa cytochrome c551. It is shown that no ligand exchange takes place at the haem in this species over the pH range 6.0-11.3.     

An Arabinogalactan-protein from the Leaves of Nicotiana tabacum

The signals of fatty acids in the form of triglycerides were observed in the ¹³C NMR spectrum of an intact soybean seed. The major fatty acid component composition of triglycerides in a soybean seed, which includes linoleic acid, oleic acid and palmitic acid, was estimated by subtracting the spectra of authentic fatty acids from the spectrum of the intact soybean seeds. The fatty acid compositions of seeds of 11 Japanese soybean cultivars and 5 lines bred at the Asian Vegetable Research and Development Center (AVRDC) were estimated by this rapid (within lhr for one seed) and nondestructive analytical method.     

Analysis of the acid and alkaline dissociation of earthworm hemoglobin, Lumbricus terrestris, by front-face fluorescence spectroscopy

The steady-state fluorescence properties of the multisubunit hemoglobin isolated from the earthworm, Lumbricus terrestris, were studied by front-face fluorometry. Acid and alkaline dissociation of this high-molecular-weight hemoglobin were examined over the pH range 3.7-12.5 using different liganded states (oxy, CO, met). The relative intensity of the emission maximum at 320 nm (exc. 280 nm) is ligand-dependent increasing as follows: oxy less than deoxy less than CO less than met at pH 7.0. The intensity of the emission maximum of oxyhemoglobin at the alkaline acid end point, pH 10.5 (333 nm), is significantly greater than that observed at the acid end point, pH 4.18 (320 nm), suggesting different subunit dissociation. The spectra of oxyhemoglobin at pH 4.18 and the spectrum of carbonmonoxy hemoglobin at pH 7.0 in the presence of 1 M magnesium chloride were almost identical, indicating similar subunit dissociation. Difference spectrum (pH 9.0-7.2) of fluorescence emission (exc. 305) resulted in a maximum at 341 nm, indicative of tyrosinate formation. This suggests that tyrosine(s) may also be located at the subunit interface(s) of this hemoglobin. These studies indicate that several aromatic amino acid residues are associated with the critical sites of subunit interactions within this molecule. Analysis of the fluorescence spectra also suggests that the formation of different subunit species resulting from acid and alkaline dissociation cannot be ruled out.     

Optimization of the determination of RNA nucleotide composition by ultraviolet spectral analysis

We have optimized the spectrophotometric method for the quantitative analysis of a tetranucleotide mixture (for example an RNA hydrolysate) by carefully choosing the wavelengths and the pH at which the absorbance measurements were made, and estimated the error made on the calculated concentration of each nucleotide. The most convenient method involved the measurement of only four absorbances (at 228 nm at pH 13; 200, 212 and 284 nm at pH 2). If the sensitivity of the spectrophotometer is 0.001 unit absorbance, the absolute error made on the calculated percentage of each nucleotide is lower than 0.33%.     

Characterization of the fluorodihydrouracil substituent in 5-fluorouracil-containing Escherichia coli transfer RNA

The fluorodihydrouridine derivative previously detected in one of two isoaccepting forms of FUra-substituted Escherichia coli tRNAMetf has been further characterized. This substituent is responsible for the 19F resonance observed 15 ppm upfield from free FUra (= 0 ppm) in the high resolution 19F-NMR spectra of FUra-substituted tRNA purified by chromatography on DEAE-cellulose, at pH 8.9, to remove normal tRNA. Similar highfield 19F signals have now been observed in the spectra of two other purified fluorinated E. coli tRNAs, tRNAMetm and tRNAVal1, as well as in unfractionated tRNA, indicating the widespread occurrence of the constituent. Comparison with 19F spectrum of the model compound 5'-deoxy-5-fluoro-5,6-dihydrouridine (dH56FUrd) (delta FUra = -31.4 ppm; JHF = 48 Hz) indicates that the substituent does not contain an intact fluorodihydrouridine ring. dH56FUrd is considerably more alkali labile than 5,6-dihydrouridine (H56Urd). At pH 8.9, where H56Urd is stable, dH56FUrd is degraded to a derivative, presumably a fluoroureidopropionic acid, with a 19F resonance at - 15.7 ppm that nearly coincides with the upfield peak in the spectrum of pH 8.9-treated tRNA. The 19F-NMR spectrum of fluorinated tRNA, not exposed to pH 8.9, exhibits two peaks 31 and 32 ppm upfield of FUra, in place of the 19F signal at - 15 ppm. Hydrolysis of this tRNA with RNAase T2 produces a sharp doublet 33 ppm upfield (JHF = 45 Hz). Similarities of the 19F chemical shift and coupling constant to those of dH56FUrd, allows assignment of the peak at -33 ppm to an intact fluorodihydrouridine residue in the tRNA. Our results demonstrate that FUra residues incorporated into E. coli tRNA at sites normally occupied by dihydrouridine can be recognized by tRNA-modifying enzymes and reduced to fluorodihydrouridine. This substituent is labile at moderately alkaline pH values and undergoes ring-opening during purification of the tRNA.     

Base composition of intact nucleic acid oligomers

The base composition of intact, purified, oligonucleotides has been determined by comparing the absorption spectrum of each oligomer with calculated curves. The spectral curve of each oligomer was measured in 7M urea at three pH values and digitized at 1-mμ intervals. The calculated curves consisted of the sums of the absorption spectra of mononucleotides in 7M urea at the same pH's. A computer was used to make the comparisons and establish the best fit. Results are presented for nine of the ten possible dimer composition isomers; trimers from pancreatic and T₁ ribonuclease hydrolyzates of TMV-RNA; poly-A, poly-C, and poly-U; and intact tobacco mosaic virus-RNA.     

Phosphorus NMR analysis of human white matter in mixed non-ionic detergent micelles

In order to study the lipid composition of human white matter, we have developed a 31P NMR spectroscopy method, which allows the determination and quantitation of the main phospholipids found in biological membranes. The technique is based upon the use of a non-ionic detergent (Triton X-100) which induces, in aqueous media, the formation of mixed micelles that are magnetically isotropic. The linewidths and chemical shifts depend on both the molar ratio detergent/phospholipid and the pH of the suspension. After determination of the optimum values for these two parameters, 31P NMR spectra were recorded, in which all phospholipid resonances were resolved. After determining precise chemical shifts for each phospholipid, concentrations were measured by comparing the peak areas with that of an internal standard. Analysis of the complex phospholipid composition of human white matter using this method gave values very close to that found in the literature for such tissue. Moreover this nondestructive method proved to be very sensitive since less than 1 mg of a mixture of phospholipids was needed.     

The secondary structure of ribosomal ribonucleic acid in solution

1. The u.v.-absorption spectrum of ribosomal RNA from rabbit reticulocytes was studied as a function of temperature at different pH values. The changes in the spectrum over the range 220-320mmu were interpreted on the basis of the assumption that the effect of denaturation and ionization are additive. The results suggest that in neutral salt solutions the secondary structure of the ribosomal RNA samples studied is due to two species of helical segments stabilized principally, if not solely, by complementary base pairs but differing in nucleotide composition: each species appears to be heterogeneous in other respects in view of the breadth of the melting ranges. 2. The number of base pairs per helical segment was estimated to be small (between 4 and 17) on the basis of the relation between melting temperature and chain length previously established by Lipsett and others for model compounds. Small fragments (about 2s) obtained by alkaline hydrolysis appeared to form the same helical segments as the intact molecule in accord with the estimated size of these segments. 3. Specific nucleotide sequences appear necessary to account for the hysteresis observed on titrating ribosomal RNA with acid or alkali within the range pH3.0-7.0 since this phenomenon was less pronounced for Escherichia coli transfer RNA and for RNA from turnip yellow-mosaic virus.     

Infrared spectra of glycosaminoglycans in deuterium oxide and deuterium chloride solution: Quantitative evaluation of uronic acid and acetamidodeoxyhexose moieties

I.r. spectra in the “carbonyl region” (1800-1400 cm^?1) have been obtained for aqueous solutions of a number of glycosaminoglycans and model compounds. In D₂O solution, the uronic carboxylate and acetamido groups absorb at characteristic frequencies (ν_asCOO^?, 1605 ±5; ν₅COO^?, 1420 ±5; Amide I, 1623 ±3; and Amide II, 1480 ±2 cm^?1). In acidic (m DCl) solution, the amide bands remain substantially unmodified, whereas those of the carboxylate anion disappear and are replaced by a single band due to the undissociated carboxyl (νCOOH, 1723 ±3 cm^?1). These bands can be used for quantitative evaluation of the uronic acid and acetamidodeoxyhexose moieties of glycosaminoglycans, using either standard polysaccharides or the corresponding monosaccharides as reference compounds. For D₂O solutions, the absorbances of the carboxylate and acetamido groups have been measured by graphical extrapolation of the corresponding most-intense bands (ν_asCOO^? and Amide I). In DCl, the two analytical bands (νCOOH and Amide I) are well-resolved, and analyses have been performed directly from the absorbances recorded. I.r. data for the uronic acid and 2-acetamido-2-deoxyhexose content of various reference samples of glycosaminoglycans are in reasonable agreement with those expected from the “established” structures, as well as with those obtained by colorimetric and conductimetric methods. When sodium d-glucuronate was used as the reference standard, the i.r. data gave relatively low values for the uronic acid content of heparin. The apparent acid dissociation constants of chondroitin 4-sulfate and heparin were estimated from the absorbance of νCOO^? and/or the νCOOH bands at different pH (pD) values.     

Circular dichroism of the complex between N-N′-diethylpseudoisocyanine and sodium poly(L-glutamate) in aqueous solutions

In the presence of sodium poly(L -glutamate) at pH = 7.5 the dye pseudoisocyanine in dilute aqueous solution (C_d = 1.10 × 10^?5 M) and low P/D values exhibits an absorption spectrum with a very sharp red-shifted J-band. Under the same conditions circular dichroism (CD) in the visible region is observed with an extremely sharp peak at the position of the J-band. At pH = 4.6, where the polypeptide is in the α-helix conformation, no such J-band is observed and no CD spectrum can be detected at the same P/D values. CD spectra in the uv range demonstrate that the occurrence of the dye–polypeptide complex which gives rise to the J-band at slightly alkaline pH is not accompanied by a conformational transition of the polypeptide towards the α-helical form.     

大豆根腐病生防菌KJB04-11的鉴定及其产生的脂肽类抗生素

从大豆根围筛选到1株对尖孢镰刀菌和立枯丝核菌都具有很好拮抗作用的菌株KJB04-11,经形态观察、生理生化特征和16SrDNA序列分析,属于枯草芽孢杆菌(Bacillussubtilis)。具有抗菌活性的KJB04-11发酵液无菌滤液对热和酸碱具有较强的稳定性。采用SephadexG-25柱层析、反相HPLC和冷冻干燥从KJB04-11发酵液中分离纯化了抗菌活性成分。由红外光谱、MALDI-TOF-MS、氨基酸组成及脂肽合成酶基因扩增结果推测该菌株产生的抗菌物质为C16、C17的mycosubtilin和C15的surfactin。田间试验表明,大豆种子经KJB04-11发酵液包衣处理对大豆根腐病防效为53.6%,大豆产量提高12.5%。     

Temperature-Induced Changes in the Sporicidal Activity and Chemical Properties of Glutaraldehyde

Freshly prepared 2% acid and alkaline glutaraldehyde solutions were stored at 4, 20, and 37 C. At intervals, samples were removed and changes in pH, ultraviolet spectrum, and sporicidal activity (against Bacillus pumilus spores) were recorded. Alkaline solutions stored at 4 C showed little changes in these properties, whereas such solutions stored at 37 C became turbid and showed a decrease in pH, marked changes in ultraviolet spectrum, and an almost complete loss of sporicidal activity. Intermediate results were obtained with alkaline solutions stored at 20 C. In contrast, acid 2% glutaraldehyde solutions (initial pH 3.5) showed comparatively few changes in their properties. Treatment of spores with freshly prepared glutaraldehyde solutions (0.5%) at temperature above 40 C reduced the effect of pH on sporicidal activity.     

1H NMR study of the solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus

The solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I) have been investigated by high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz. The pH dependence of the spectra has been examined over the range 1.1-12.2 at 27 degrees C. Individual pKa values have been obtained for the alpha-ammonium group of Ala-1 (8.6) and the side chains of Glu-8 (3.7), Tyr-36 (10.9), and Tyr-37 (10.8). For the remaining seven carboxyl groups in the molecule (from five Asp, Glu-31, and the C-terminus), four pKa values, viz., 2.8, 3.5, 4.1 and 6.4, can be clearly identified. The five Lys residues titrate in the range 10.5-11, but individual pKa values could not be obtained because of peak overlap. Conformational changes associated with the protonation of carboxylates occur below pH 4, while in the alkaline pH range major unfolding occurs above pH 10. The molecule also unfolds at elevated temperatures, having a transition temperature of ca. 55 degrees C at pH 5.25. Exchange of the backbone amide protons has been monitored at various values of pH and temperature in the ranges pH 4-5 and 12-27 degrees C. Up to 18 slowly exchanging amides are observed, consistent with the existence of a core of hydrogen-bonded secondary structure, most probably beta-sheet.(ABSTRACT TRUNCATED AT 250 WORDS)