Effect of Sodium Nitrite on the Destruction of Thiamine

The effect of sodium nitrite on the destruction of thiamine was investigated. When sodium nitrite-containing thiamine solution was treated by the condition of heating at 75°C for 60 min, elemental sulfur and 4-methyl-5-(β-hydroxyethyl) thiazole were identified, and thiochrome was estimated. When sodium nitrite-free thiamine solution was heated at 75°C for 60 min, 4-methyl-5-(β-hydroxyethyl) thiazole was a main product, and elemental sulfur and thiochrome were not produced. From these results, it showed that elemental sulfur and thiochrome were produced from thiamine by the effect of sodium nitrite.     

Oxidation of thiamine on reaction with nitrogen dioxide generated by ferric myoglobin and hemoglobin in the presence of nitrite and hydrogen peroxide

It is shown that nitrogen dioxide oxidizes thiamine to thiamine disulfide, thiochrome, and oxodihydrothiochrome (ODTch). The latter is formed during oxidation of thiochrome by nitrogen dioxide. Nitrogen dioxide was produced by incubation of nitrite with horse ferric myoglobin and human hemoglobin in the presence of hydrogen peroxide. After addition of tyrosine or phenol to aqueous solutions containing oxoferryl forms of the hemoproteins, thiamine, and nitrite, the yield of thiochrome greatly increased, whereas the yield of ODTch decreased. In the presence of high concentrations of tyrosine or phenol compounds ODTch was not formed at all. The neutral form of thiamine with the closed thiazole cycle and minor tricyclic form of thiamine do not enter the heme pocket of the protein and do not interact with the oxoferryl heme complex Fe(IV=O) or porphyrin radical. The tricyclic form of thiamine is oxidized to thiochrome by tyrosyl radicals located on the surface of the hemoprotein. The thiol form of thiamine is oxidized to thiamine disulfide by both hemoprotein tyrosyl radicals and oxoferryl heme complexes. Nitrite and also tyrosine, tyramine, and phenol readily penetrate into the heme pocket of the protein and reduce the oxyferryl complex to ferric cation. These reactions yield nitrogen dioxide as well as tyrosyl and phenoxyl radicals of tyrosine molecules and phenol compounds, respectively. Tyrosyl and phenoxyl radicals of low molecular weight compounds oxidize thiamine only to thiochrome and thiamine disulfide. The effect of oxoferryl forms of myoglobin and hemoglobin, nitrogen dioxide, and phenol on thiamine oxidative transformation as well as antioxidant properties of the hydrophobic thiamine metabolites thiochrome and ODTch are discussed.     

利用亚硝酸钠选育法夫酵母虾青素高产菌株

以亚硝酸钠作为筛选剂选择性分离法夫酵母虾青素高产菌株。实验研究表明,在亚硝酸钠存在的情况下,法夫酵母的生长和虾青素合成量均会减少;当亚硝酸钠浓度为5000μmol/L时,法夫酵母的致死率为100%。挑取200株经过甲基磺酸乙酯（EMS）诱变后的法夫酵母,以5000μmol/L的亚硝酸钠为筛选剂摇瓶发酵后测得虾青素体积产率为正突变的菌株有87株,正突变率为43.5%。挑取其中8株进行复筛,编号为N030的菌株比出发菌株的虾青素体积产率和细胞产率分别提高了39.3%和89.3%。结果说明,亚硝酸钠可作为法夫酵母虾青素高产菌株的筛选剂,用于提高菌种的筛选效率。     

A fluorescent assay for proteolytic enzymes

A method is described which permits the assay of proteolytic enzyme activity on protein substrates without precipitation or filtration steps, utilizing a fluorescent reagent which is specific for primary amines. The assay is about 100 times more sensitive than the Lowry method, much faster and less complicated. Ambiguities concerning the absorbing species are largely eliminated. The reagent (Fluorescamine, Hoffmann-La Roche RO-20-7234) yields fluorescent compounds with amino acids at pH 9.0 and with peptides at pH 6.8, but possesses no fluorescence by itself.     

A continuous fluorescence assay for protein kinase C

A 6-acryloyl-2-dimethylaminonapthalene (acrylodan)-labeled 25-amino acid peptide (acrylodan-CKK-KKRFSFKKSFKLSGFSFKKNKK-COO-), containing the protein kinase C (PKC) phosphorylation sites of brain myristoylated alanine-rich kinase C substrate protein, undergoes a 20% fluorescence decrease when it is phosphorylated by phospholipid/calcium-dependent protein kinase (PKC). This fluorescence decrease is dependent on the presence of PKC, calcium (half-maximal stimulation at pCa = 6.2), phosphatidylserine, diacylglycerol, or phorbol-12-myristate-13-acetate (half-maximal stimulation at 2 nM) and ATP, and correlates well (r = 0.997) with [32P]phosphate incorporation into the peptide. This fluorescence assay allows detection of 0.02 nM PKC, while similar concentrations of cyclic AMP-dependent or type II calmodulin-dependent protein kinases produced no change in peptide fluorescence. The method can be used to assay purified PKC as well as activity in crude brain homogenates. Incubation of PKC with staurosporine inhibits the fluorescence decrease with an IC50 of 2 nM. Thus the fluorescence decrease that occurs in the acrylodan-peptide provides a continuous fluorescence assay for PKC activity.     

A new pitfall in detecting biological end products of nitric oxide-nitration, nitros(yl)ation and nitrite/nitrate artefacts during freezing.

The present study shows that when freezing nitrite containing biological samples in the presence of sodium and phosphate, a process of tyrosine nitration and S-nitrosocysteine formation is observed. The underlying mechanism is obviously based on the already described pH decrease in sodium phosphate buffered solutions during the freezing process and probably involves nitrous acid as an intermediate. However, in pure potassium phosphate buffer freeze-artefacts were absent. The yield of 3-nitrotyrosine from albumin-bound or free tyrosine depends not only on the concentration of nitrite, tyrosine or protein, and sodium phosphate but also on the velocity of the freezing process. Nitrite and nitrate were quantified by the Griess/nitrate reductase assay. 3-nitrotyrosine formation was quantitatively measured by HPLC analysis with optical and electrochemical detection as well as qualitatively investigated by immunohistochemistry and slot blot analysis using 3-nitrotyrosine specific antibodies. The formation of S-nitrosocysteine was detected by S-nitrosothiol specific antibodies and quantified by a fluorometric assay. Irrespective of the mechanism and although the here presented results cannot be generalized, the data warrant caution for the analysis of nitration or nitros(yl)ation products following freezing of nitrite containing biological material.     

Removal of sodium channel inactivation in squid giant axons by n-bromoacetamide

The group-specific protein reagents, N-bromacetamide (NBA) and N- bromosuccinimide (NBS), modify sodium channel gating when perfused inside squid axons. The normal fast inactivation of sodium channels is irreversibly destroyed by 1 mM NBA or NBS near neutral pH. NBA apparently exhibits an all-or-none destruction of the inactivation process at the single channel level in a manner similar to internal perfusion of Pronase. Despite the complete removal of inactivation by NBA, the voltage-dependent activation of sodium channels remains unaltered as determined by (a) sodium current turn-on kinetics, (b) sodium tail current kinetics, (c) voltage dependence of steady-state activation, and (d) sensitivity of sodium channels to external calcium concentration. NBA and NBS, which can cleave peptide bonds only at tryptophan, tyrosine, or histidine residues and can oxidize sulfur- containing amino acids, were directly compared with regard to effects on sodium inactivation to several other reagents exhibiting overlapping protein reactivity spectra. N-acetylimidazole, a tyrosine-specific reagent, was the only other compound examined capable of partially mimicking NBA. Our results are consistent with recent models of sodium inactivation and support the involvement of a tyrosine residue in the inactivation gating structure of the sodium channel.     

Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine

Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure.     

Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins

A sensitive quantitative method has been developed to determine the number of disulfide bonds in peptides and proteins. The disulfide bonds of several peptides and proteins were cleaved quantitatively by excess sodium sulfite at pH 9.5 and room temperature. Guanidine thiocyanate (2 M) was added to the protein solutions in order to denature them and thereby make the disulfide bonds accessible. The reaction with sulfite leads to a thiosulfonate and a free sulfhydryl group; the concentration of the latter was determined by reaction with disodium 2-nitro-5-thiosulfobenzoate (NTSB) in the presence of excess sodium sulfite. The synthesis, purification, and characterization of NTSB are described. The assay is rapid, requiring 3-5 min for oligopeptides and 20 min for proteins, and is as sensitive and quantitative as the sulfhydryl group assay employing 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). It can be used for the analysis of as little as 10(8) mol of disulfide bonds, with an error of +/- 3%.     

Quantitative filtration-blotting of protein in the presence of sodium dodecyl sulfate and its use for protein assay

It is thought that sodium dodecyl sulfate (SDS), an anionic detergent, binds to hydrophobic moieties of peptide to destroy the conformational structure of protein. Because of this property, it is involved in many biochemical procedures such as separations of protein and proteolytic digestion. In the course of our study on a solid-phase protein assay, we found that SDS acts as an effective reagent for protein blotting onto a hydrophobic membrane of polyvinylidene difluoride with a manifold dot-blot apparatus. At least 0.1% SDS in an acid-ethanol blotting solution, while reducing the bias of pronounced interferers for protein assay to protein-membrane interaction, quantitatively retains protein on the membrane. Presumably, protein denatures by SDS to become an unfolded state and adsorbs into the membrane by hydrophobic interaction, even in the presence of excess SDS. Therefore, bolts stained with a pyrogallol red-molybdate complex (Pyromolex) reagent unreactive to the membrane allowed a precise protein determination without significant interference of materials, especially detergents in the sample solution. The filtration-blotting with SDS would be a crucial procedure for quantitative analyses such as immunoblotting in detergent-containing samples, together with the solid-phase protein assay with limited sample volumes, such as 20 microL or less.     

Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction

We report herein the development of an efficient fluorescence assay for serine/threonine kinases using a peptide array. Our approach is based on chemical reactions specific to phosphoserine and phosphothreonine residues, that is, base-mediated beta-elimination of the phosphate group and subsequent Michael addition of a thiol-containing fluorescent reagent. This procedure enables the covalent introduction of a fluorescent moiety into the phosphorylated peptide. Novel fluorescent reagents were designed for this purpose and synthesized. With these reagents, protein kinase A (PKA) and Akt-1 activities were readily detected. Our method can also be used to measure the activity of kinase inhibitors. This assay is expected to be widely applicable in kinase research.     

Enzyme assay for protein kinase using micellar electrokinetic chromatography with laser-induced fluorescence detection

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection has been developed for a protein kinase assay. This protein kinase assay could readily determine the phosphorylation activity of substrate peptide kemptide using cAMP-dependent protein kinase (PKA) as a model enzyme. Kemptide and phosphorylated kemptide could be reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent for LIF detection by directly adding NBD-F into the PKA enzymatic reaction mixture. These derivatives of substrate and product were separated and detected within the analysis time of 5 min by micellar electrokinetic mode using a mixture of sodium dodecylsulfate and methanol as a running buffer. Good linearity of the peak response of the phosphorylated kemptide was obtained over the range of 1-20 mU/tube of PKA in the assay. The relative standard deviation of the peak areas of the phosphorylated kemptide using 2, 5 and 10 mU/tube of PKA were calculated to <10.4%, indicating that the assay was reproducible. Also, IC(50) values of six PKA inhibitors, the K(i) value and the inhibition pattern of one inhibitor, which were calculated to estimate by the variation of the peak area of the phosphorylated kemptide using 5 mU/tube of PKA, were consistent with the published data. The sensitivity of the assay was higher than that of enzyme-linked immunosorbent assay (ELISA) for PKA phosphorylation activity, as IC(50) values, K(i) value, and the inhibition mechanism of inhibitors could be estimated using one-tenth amounts of PKA, compared with that of ELISA. The MEKC-LIF is expected to be very useful for protein kinase assay and its application to the estimation of inhibitors because this method does not entail experimentally troublesome procedures such as the preparation of antibody or fluorescence-labeled substrate.     

A homogeneous time-resolved fluorescence resonance energy transfer assay for phosphatidylserine exposure on apoptotic cells

A simple, “mix-and-measure” microplate assay for phosphatidylserine (PtdSer) exposure on the surface of apoptotic cells is described. The assay exploits the fact that annexin V, a protein with high affinity and specificity for PtdSer, forms trimers and higher order oligomers on binding to membranes containing PtdSer. The transition from soluble monomer to cell-bound oligomer is detected using time-resolved fluorescence resonance energy transfer from europium chelate-labeled annexin V to Cy5-labeled annexin V. PtdSer detection is achieved by a single addition of a reagent mix containing labeled annexins and calcium ions directly to cell cultures in a 96-well plate, followed by a brief incubation before fluorescence measurement. The assay can be used to quantify PtdSer exposure on both suspension cells and adherent cells in situ. This method is simpler and faster than existing annexin V binding assays based on flow cytometry or microscopy, and it yields precise data with Z’ values of 0.6-0.7.     

Nitrite-assisted peptide iodination and conjugation.

In this study, a simple method for selective iodination of peptides and proteins is established. Using angiotensin II as the model system, we demonstrate that nitrite catalyzed the selective iodination of the peptide at the N-terminus in an acidic solution. The N-terminal-iodinated peptides alkylated thiol-containing molecules such as N-acetylcysteine and glutathione to form peptide conjugates in a basic solution. Reactive species formed by increasing the pH of the reaction mixture of sodium nitrite and sodium iodide from 4 to 8 selectively iodinated peptides and proteins at tyrosine and histidine residues. These results show that nitrite is a useful catalyst for peptide and protein ligation.     

Formation of the cystine between cysteine 225 and cysteine 462 from ribonucleoside diphosphate reductase is kinetically competent

Participation of the formation of the cystine between cysteine 225 and cysteine 462 in the R1 protein to the enzymatic mechanism of aerobic ribonucleoside diphosphate reductase from Escherichia coli has been examined by use of rapid quenching and site-directed immunochemistry. Prereduced ribonucleotide reductase in the presence of ATP was mixed with CDP in a quench flow apparatus. The reaction was terminated with a solution of acetic acid and N-ethylmaleimide. The protein was precipitated and digested with chymotrypsin and the proteinase from Staphylococcus aureus strain V8 in the presence of N-ethylmaleimide to yield the peptide SS[S-(N-ethylsuccinimid-2-yl)cysteinyl]VLIE containing cysteine 225 and the mixed disulfide between the peptide SSCVLIE and the peptide IALCTL containing cysteine 462. These two peptides were retrieved together from the digest by immunoadsorption. The affinity-purified peptides were modified at their amino termini with the fluorescent reagent 6-aminoquinolyl-N-hydroxysuccimidyl carbamate and submitted to high-pressure liquid chromatography. The areas of the respective peaks of fluorescence corresponding to the S-(N-ethylsuccimidyl) peptide, and the mixed disulfide were used to determine the percentage of the cystine that had formed during each interval. The rate constant for the formation of the cystine following the association of free, fully reduced ribonucleotide reductase with the reactant CDP was 8 s(-)(1). Because only 50% of the active sites participated in this pre-steady-state reaction, the maximum steady-state rate consistent with the involvement of this cystine in the enzymatic reaction would be 4 s(-1). Since the turnover number of the enzyme under the same conditions in a steady state assay was only 1 s(-)(1), the formation of the cystine between these two cysteines is kinetically competent.     

Modification of albumin with different degrees of the oxidation of SH-groups in the reaction with glucose

A molecule of the major blood protein albumin contains 34 cysteine residues involved in disulfide bonds and one unpaired SH-group of residue Cys34. Normally, 20–30% of these SH-groups are oxidized and form disulfide bonds or the derivatives of sulfenic, sulfinic, and sulfonic acids. The goal of the present work was to study the influence of the degree of oxidation of sulfhydryl groups on the capacity of albumin for glycation. Commercially available human albumin containing 0.4 moles of sulfhydryl groups per 1 mole of the protein (nonmercaptalbumin) was used. Disulfide bonds in this preparation were reduced with dithiothreitol to 0.7 mole/mole to give mercaptalbumin. The preparations were incubated for three weeks with glucose at a concentration of 5 and 50 mM. The content of ketoamine, a glycation product, was determined by the colorimetric method, the content of pentosidine (glycation end product) was analyzed by fluorescence, and the content of SH-groups was determined using the Ellman’s reagent. Changes in the structure and properties of the protein during glycation were studied by fluorescence and HPLC. During the incubation of both albumin preparations with 5 mM glucose, no significant increase in the ketoamine content was observed, whereas the incubation with 50 mM glucose was accompanied by a considerable accumulation of ketoamine. It was found that the greatest amount of ketoamine under these conditions forms in nonmercaptalbumin; in this case, the intensity of tryptophan fluorescence decreases. The intensity of pentosidine fluorescence increases with increasing content of ketoamine. The results obtained enable the conclusion that the oxidation of free SH-groups of the protein changes its conformation; as a result, the glycation of earlier hidden sites becomes possible, and the degree of protein glycation increases.     

Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A

A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.     

Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography

Various analytical techniques have been developed to determine nitrite and nitrate, oxidation metabolites of nitric oxide (NO), in biological samples. HPLC is a widely used method to quantify these two anions in plasma, serum, urine, saliva, cerebrospinal fluid, tissue extracts, and fetal fluids, as well as meats and cell culture medium. The detection principles include UV and VIS absorbance, electrochemistry, chemiluminescence, and fluorescence. UV or VIS absorbance and electrochemistry allow simultaneous detection of nitrite and nitrate but are vulnerable to the severe interference from chloride present in biological samples. Chemiluminescence and fluorescence detection improve the assay sensitivity and are unaffected by chloride but cannot be applied to a simultaneous analysis of nitrite and nitrate. The choice of a detection method largely depends on sample type and facility availability. The recently developed fluorometric HPLC method, which involves pre-column derivatization of nitrite with 2,3-diaminonaphthalene (DAN) and the enzymatic conversion of nitrate into nitrite, offers the advantages of easy sample preparation, simple derivatization, stable fluorescent derivatives, rapid analysis, high sensitivity and specificity, lack of interferences, and easy automation for determining nitrite and nitrate in all biological samples including cell culture medium. To ensure accurate analysis, care should be taken in sample collection, processing, and derivatization as well as preparation of reagent solutions and mobile phases, to prevent environmental contamination. HPLC methods provide a useful research tool for studying NO biochemistry, physiology and pharmacology.     

A fluorescence lifetime-based assay for serine and threonine kinases that is suitable for high-throughput screening

We describe the development of a novel method for the assay of serine/threonine protein kinases based on fluorescence lifetime. The assay consists of three generic peptides (which have been used by others in the assay of >140 protein kinases in various assay formats) labeled with a long lifetime fluorescent dye (14 or 17 ns) that act as substrates for protein kinases and an iron(III) chelate that modulates the fluorescence lifetime of the peptide only when it is phosphorylated. The decrease in average fluorescence lifetime as measured in a recently developed fluorescence lifetime plate reader (Edinburgh Instruments) is a measure of the degree of phosphorylation of the peptide. We present data showing that the assay performs as well as, and in some cases better than, the “gold standard” radiometric kinase assays with respect to Z′ values, demonstrating its utility in high-throughput screening applications. We also show that the assay gives nearly identical results in trial screening to those obtained by radiometric assays and that it is less prone to interference than simple fluorescence intensity measurements.     

Tandem Peptide Ligation for Synthetic and Natural Biologicals

We describe the concept and methods of peptide ligation and tandem peptide ligation for preparing synthetic and natural biologicals. Peptide ligation is a segment coupling method for free peptides or proteins through an amide bond without the use of a coupling reagent or a protecting group scheme. Because unprotected peptides or proteins prepared from either a chemical or biochemical source are being used as building blocks, the ligation removes the size limitation for peptide and protein synthesis. A key feature of the peptide ligation is that the coupling reaction is orthogonal, i.e. it is specific to a particular alpha-amino terminus (NT). This NT-amino acid-specific feature permits the development of a tandem peptide ligation method employing three unprotected peptide segments containing different NT-amino acids to form consecutively two amide bonds, an Xaa-SPro (thiaproline) and then an Xaa-Cys. This strategy was tested in peptides ranging from 28 to 70 amino acid residues, including analogues of somatostatins and two CC-chemokines MIP-1alpha and MIP-1beta. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for preparing protein biologicals and synthetic vaccines.