Amino acid end-group in commercial heparin. II. Reaction kinetics of the amino end groups with 2,4,6-trinitrobenzenesulfonic acid

Three different commercial heparins were trinitophenylated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) under aqueous conditions. The reaction kinetics of amino groups in heparin with TNBS showed that the reactivities of amino groups were significantly different for free amino groups on heparin, compared to reactivities in peptides and amino acid residues attached to heparin molecules. With TNBS, unreactive amino groups were always present during the reaction.     

Amino acid end-group in commercial heparin. III. Determination of the number of amino groups of amino acid and hexosamine residues per heparin molecule

The reactions of heparin with 2,4,6-trinitrobenzenesulfonic acid (TNBS) were studied spectrometrically. Seven different commercial heparins were used in this study. The amino groups react with TNBS to form equimolar amounts of trinitrophenylated (TNP) amino groups and bisulfite ions. The TNP-amino groups further react with bisulfite ions to form the monosubstituted anionic sigma complex. The absorption spectrum with two maxima at approximately 350 nm and approximately 420 nm, characteristic of either the TNP-amino groups or the complex, was analyzed for the reaction of TNBS with heparin. It was shown that the reactivities of TNBS with amino groups from α-amino acid and hexosamine residues are greatly different. By combining the results of the reaction kinetics and the reaction of heparin with Sanger's reagent, the number of the α-amino groups and the free amino groups in hexosamine residues were determined. These data have been performed with a range of heparins from different commercial sources, of different activities and physical characteristics. No correlation was found between the free amino contents of these heparins and biological potency. © 1993 John Wiley & Sons, Inc.     

Phospholipase D modified with a polyethylene glycol derivative

Phospholipase D from Streptomyces sp. AA586, PLDP, was modified with methoxypolyethylene glycol succinimidylsuccinate (ss-PEG), an active derivative of polyethylene glycol. By titration with trinitrobenzene sulfonate (TNBS), approximately 70% of the free amino groups in the enzyme protein were shown to be modified by treatment with ss-PEG. By this modification, the molecular weight of the enzyme was increased, judging from the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration with Toyopearl HW-55F and TNBS titration. Due to loss of cationic charges, the enzyme protein became eluted faster in high performance liquid chromatography with CM-Toyopearl. By modification with ss-PEG, the enzyme became fairly thermostable, while pH-stability and optimal pH were not influenced. The value of Km for phosphatidylcholine of the hydrolytic reaction increased 2-fold, whereas that of the transphosphatidyl reaction was not significantly altered.     

Sample clean-up method for analysis of complex-type N-glycans released from glycopeptides

N-Glycans in glycoprotein can be liberated either from glycoproteins or from their glycopeptides with glycoamidases. The latter approach is preferable, because it requires a smaller amount of the enzyme, and yields N-glycans in excellent yields. Moreover it alleviates the necessity of removing from the reaction mixture the detergents needed to denature the glycoproteins. On the other hand, this approach necessitates removal of interfering peptidic materials, because some of the peptide peaks often overlap with the peaks of carbohydrate chains in high-performance anion-exchange chromatography (HPAEC). These peptidic materials also hinder labeling of N-glycans by reductive amination. We have tried to remove the interfering peptidic materials by several different methods--octadecyl (C18) silica cartridge, cation-exchange resin column, and graphitized carbon cartridge. Unfortunately, none of these could completely remove the interfering peptidic materials. Therefore, we resorted to modify the amino groups of the peptidic materials with sodium 2,4,6-trinitro-benzene-1-sulfonate (TNBS) to render them more hydrophobic, so that they can be retained more strongly on the C18 or graphitized carbon cartridges. In the model study presented here, we were able to obtain N-glycans for HPAEC analyses without any interfering materials by a combination of TNBS reaction and graphitized carbon treatment.     

Immobilization of the restriction endonuclease PstI

PstI has been immobilized in agarose. A solution of low melting agarose containing 1,6-hexamethylenediamine and PstI formed a gel that was effective in the linearization of pBR322 DNA. The gel containing PstI could be treated with 1,5-bis(N-acetylamino-N-succinimidoxy carbonyl)pentane, a crosslinking agent, without affecting the enzyme activity. Polymerization of acrylamide in presence of PstI led to conisderably reduced enzyme activity, although EcoRI under identical conditions showed high activity. It was found that acetylation of amino groups in PstI, by reaction with hydroxysuccinimide acetate, led to total inactivation of the enzyme activity. This reaction showed the presence of reactive amino groups that were essential for the enzyme activity of PstI. Involvement of these amino groups in binding to activated Sepharose 4B, during covalent immobilization, was responsible for inactive enzyme preparations.     

Studies on the Chemical Modification of the Essential Groups of <Emphasis Type="Italic">N</Emphasis>-Acetyl-β-<Emphasis Type="SmallCaps">d</Emphasis>-Glucosaminidase from Viscera of Green Crab (<Emphasis Type="Italic">Scylla Serrata</Emphasis>)

The chemical modification of N-acetyl-β-d-glucosaminidase (EC3.2.1.30) from viscera of green crab (Scylla serrata) has been first studied. The modification of indole groups of tryptophan of the enzyme by N-bromosuccinimide can lead to complete inactivation, accompanying the absorption decreasing at 275 nm and the fluorescence intensity quenching at 338 nm, indicating that tryptophan is essential residue to the enzyme. The modification of histidine residue, the carboxyl groups, and lysine residue inactivates the enzyme completely or incompletely. The results show that imidazole groups of histidine residue or sulfhydryl residues, the carboxyl groups of acidic amino acid, amino groups of lysine residue, and indole groups of tryptophan were essential for the catalytic activity of enzyme, while the results demonstrate that the disulfide bonds and the carbamidine groups of arginine residues are not essential to the enzyme’s function.     

Stability and acidity constants for ternary ligand-zinc-hydroxo complexes of tetradentate tripodal ligands

Four series of tetradentate tripodal ligands containing pyridyl, 2-imidazolyl, 4-imidazolyl, amino, and/or carboxylic groups were synthesized as hydrolytic zinc enzyme models in order to elucidate the effect of various coordination environments on zinc binding and the acidity of zinc-bound water. In aqueous solution, ligands with same charges showed a good correlation between zinc binding (log K(ZnL)) and zinc-bound water acidity (pK(a) of LZnOH(2)); the stronger the zinc binding, the higher the pK(a). The zinc-bound water acidity decreased as pyridyl groups were replaced by carboxylate groups. However, exchanging amino groups for carboxylate groups gave no change in zinc-bound water acidity regardless of the charge of the atoms in the inner coordination sphere of the metal ion. The results are consistent with the conventional notion that negatively charged carboxylate groups inherently increase zinc binding and result in decreasing zinc-bound water acidity, but also suggest that environmental effects may modulate or dominate control of acidity.     

Purification and Characterization of 3-Ketovalidoxylamine A C-N Lyase Produced by Stenotrophomonas maltrophilia

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH?7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0–10.5. The optimal temperature was found to be near 40?°C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca²⁺. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K _m value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.     

Discovery of a substrate selectivity switch in tyrosine ammonia-lyase, a member of the aromatic amino acid lyase family

Tyrosine ammonia-lyase (TAL) is a recently described member of the aromatic amino acid lyase family, which also includes phenylalanine (PAL) and histidine ammonia-lyases (HAL). TAL is highly selective for L-tyrosine, and synthesizes 4-coumaric acid as a protein cofactor or antibiotic precursor in microorganisms. In this report, we identify a single active site residue important for substrate selection in this enzyme family. Replacing the active site residue His89 with Phe in TAL completely switched its substrate selectivity from tyrosine to phenylalanine, thereby converting it into a highly active PAL. When a corresponding mutation was made in PAL, the enzyme lost PAL activity and gained TAL activity. The discovered substrate selectivity switch is a rare example of a complete alteration of substrate specificity by a single point mutation. We also show that the identity of the amino acid at the switch position can serve as a guide to predict substrate specificities of annotated aromatic amino acid lyases in genome sequences.     

Chromatographic analysis of the trinitrophenyl derivatives of insulin

The insulin molecule was derivatised by reaction with trinitrobenzenesulphonic acid (TNBS), which is known to react predominantly with free primary amino groups. The products of the reaction were analysed by reversed-phase chromatography and by further derivatisation with dansyl chloride. Under the conditions of these experiments, TNBS was found to react preferentially with glycine at position A1. This finding is discussed in terms of the tertiary structure and immunogenicity of this derivative.     

STUDIES ON THE in vitro O2-DEPENDENT INACTIVATION OF NADH-GLUTAMATE SYNTHASE FROM Chlamydomonas reinhardii STIMULATED BY FLAVINS

The NADH-glutamate synthase (EC 1.4.1.14) complex from Chlamydomonas reinhardii may experience in vitro two kinds of 0₂-dependent inactivation stimulated by flavins.
A peroxide-mediated inactivation of enzyme, which affects the NADH- and MVt-glutamate synthase activities of the complex, but not the NADH-dehydrogenase activity, can be obtained by aerobic incubation of the enzyme with NAD(P)H plus flavin. This inactivation of enzyme seems to be due to a permanent modification of sulfhydryl groups near the active site for L-glutamine or 2-oxoglutarate. The addition of 10 mM dithioerythritol to inactive NADH-glutamate synthase produces a significant, but not complete, reactivation of the enzyme.
On the other hand, the NADH-glutamate synthase is highly susceptible to a photodynamic inactivation caused by singlet 0₂. Aerobic incubation of the active enzyme with flavin under illumination leads to the irreversible inactivation of all the activities associated with the enzymatic complex.     

Surfaces Coated with Polymer Brushes Work as Carriers for Histidine Ammonia Lyase

The immobilization of enzymes on solid supports is an important challenge in biotechnology and biomedicine. In contrast to other methods, enzyme deposition in polymer brushes offers the benefit of high protein loading that preserves enzymatic activity in part due to the hydrated 3D environment that is available within the brush structure. The authors equipped planar and colloidal silica surfaces with poly(2-(diethylamino)ethyl methacrylate)-based brushes to immobilize Thermoplasma acidophilum histidine ammonia lyase, and analyzed the amount and activity of the immobilized enzyme. The poly(2-(diethylamino)ethyl methacrylate) brushes are attached to the solid silica supports either via a “grafting-to” or a “grafting-from” method. It is found that the grafting-from method results in higher amounts of deposited polymer and, consequently, higher amounts of Thermoplasma acidophilum histidine ammonia lyase. All polymer brush-modified surfaces show preserved catalytic activity of the deposited Thermoplasma acidophilum histidine ammonia lyase. However, immobilizing the enzyme in polymer brushes using the grafting-from method resulted in twice the enzymatic activity from the grafting-to approach, illustrating a successful enzyme deposition on a solid support.     

Methodology for determining degree of hydrolysis of proteins in Hydrolysates: a review

Degree of hydrolysis (DH) is defined as the proportion of cleaved peptide bonds in a protein hydrolysate. Several methods exist for determining DH; the most commonly used of these include the pH-stat, trinitrobenzenesulfonic acid (TNBS), o-phthaldialdehyde (OPA), trichloroacetic acid soluble nitrogen (SN-TCA), and formol titration methods. The pH-stat method is based on the number of protons released during hydrolysis; the TNBS, OPA, and formol titration methods are based on the measurement of amino groups generated from hydrolysis. The SN-TCA method measures the amount of TCA-soluble nitrogen, rather than DH. The pH-stat is the simplest and most commonly used method, but does not determine peptide bonds directly. In addition, the accuracy of the method depends on the type of hydrolytic enzymes used, the size of the hydrolyzed peptides, and the reaction temperature. Generally, the TNBS and OPA methods compare well and do directly determine DH. However, the assumption that the response factor for all derivatized N-terminal amino acids is similar may lead to inaccuracies. In conclusion, there is no consensus as to the best method for determining the DH of protein hydrolysates; consequently, there is a need for a standardized approach if interstudy comparisons are to be made.     

Theoretical investigation on the dissociation of (R)‐benzoin catalyzed by benzaldehyde lyase

Benzaldehyde lyase (BAL) is a versatile thiamin diphosphate (THDP)‐dependent enzyme with widespread synthetic applications in industry. Besides lyase activity, BAL also performs the functions as carboligase and decarboxylase. Unlike many other THDP‐dependent enzymes, the active center of BAL is devoid of any acid‐base amino acid residues except Glu50 and His29, and therefore, the catalytic mechanism of BAL is unusual. In this article, the dissociation mechanism of (R)‐benzoin to benzaldehyde catalyzed by BAL has been studied by using density functional theory method. The calculation results indicate that the whole reaction consists of four elementary steps, and at least two steps contribute to rate‐limiting. A big difference with other THDP‐dependent enzymes is that, in the first stage of the reaction, the ligation of substrate and THDP ylide is not companied by proton transfer, and in the subsequent transition states and intermediates, the carbonyl oxygen always exists in the form of anion. Gln113, His29, and 4′‐amino group of THDP are found to have the function to stabilize the transition states and intermediates. His29 acts as the proton acceptor in step 2 and proton donor in step 3 using one water molecule as mediator. © 2013 Wiley Periodicals, Inc.     

A turn-on fluorescent indicator for citrate with micromolar sensitivity

A turn-on fluorescent indicator for citric acid (citrate) has been developed, displaying high emission enhancement (+1500%) and low interference by other carboxylates. The sensor is based on the non-emissive copper(II) complex of a fluorescent amino amide, which, upon addition of citrate decomplexates to yield the emissive ligand. The detection limit estimated for this new chemosensing system is about 0.5 microM. This novel approach to the analysis of citrate constitutes an alternative ca. 10(2)-10(3) times more sensitive than the standard method based on the enzyme citrate lyase.     

Application of log D for the prediction of hydrophobicity in the advanced Marfey's method

In the advanced Marfey's method, the resolution between the diastereomers derivatized with 1-fluoro-2,4-dinitrophenyl-5-l-leucinamide (l-FDLA) and 1-fluoro-2,4-dinitrophenyl-5-d-leucinamide (d-FDLA) is reflected by the difference of hydrophobicity of the two functional groups at the asymmetric carbon. However, no effective method has been developed for the estimation of hydrophobicity so far. For this purpose, we introduced log D from the ACD Labs LogD and applied it to relatively simple primary amines, amino acids and secondary alcohols in the present study. It was found that the difference of the retention times (Δt_R) correlated with that of log D (Δlog D) for both diastereomers based on the obtained experimental results. Based on these results, the following procedure was proposed for the non-empirical determination of the absolute configuration of primary amines including amino acids and secondary alcohols: (1) estimate the hydrophobicity by the calculation of log D for the two substituent groups at the asymmetric carbon, (2) locate the trans-type arrangement of the two more hydrophobic substituents in the l-DLA derivative and judge the asymmetric carbon to be R or S in the trans-type that is eluted first, (3) derivatize the desired compound with l- or d-FDLA and analyze by LC/MS, and (4) compare the elution order with the prospective one and determine the absolute configuration at the asymmetric carbon. Furthermore, log D could also be used to predict the retention times of unavailable amino acids and small peptides, indicating that the combination of the advanced Marfey's method with log D would provide more reliable structural information on a mixture composed of amino acids and small peptides. The developed method is being applied to more complicated compounds.     

ROLE OF THE TRYPTOPHAN FLUORESCENT STATE IN THE ULTRAVIOLET-INDUCED INACTIVATION OF β-TRYPSIN*

Abstract— Stern-Volmer quenching constants for β-trypsin at pH 3 were determined for fluorescence quenching by histidine, acrylamide, and nitrate ion. A modified Stern-Volmer plot (Lehrer, 1971) was employed to show that all of the fluorescent tryptophanyl residues of β-trypsin were equally susceptible to quenching by acrylamide at pH 3 when the enzyme was either in its native conformation or denatured in 6 M guanidine hydrochloride (GuHCl). Fluorescence lifetime measurements indicated that acrylamide quenched β-trypsin fluorescence by a purely collisional mechanism. Solvation of tryptophanyl residues of the protein was maximal at 2.5 M GuHCl, as monitored by fluorescence emission wavelength.
Investigations of the ultraviolet-induced inactivation of β-trypsin at 295 nm were performed in the presence of acrylamide at pH 3. The quantum yields for enzyme inactivation and indole destruction (determined using the PDAB reagent) were unchanged upon depopulation of the fluorescent state by 65 per cent, whether the enzyme was in its native conformation or denatured by 6 M GuHCl. It is concluded that the fluorescent state of tryptophanyl residues of β-trypsin is not involved in enzyme inactivation or tryptophan destruction.     

The reaction of glutamic acid and trinitrobenzenesulfonic acid--kinetic study and analytical application

Some kinetic aspects of the reaction of glutamic acid (GLU) and Trinitrobenzenesulfonic acid (TNBS) were studied spectrophotometrically (420 nm), under pseudo-first order reaction conditions. The effect of GLU, TNBS, SO(2-)(3) and H(+) had been investigated. Working curves for the initial rates IR versus [GLU] are linear in the range 7.5-30.0 mM. The regression equation is IR = (8 +/- 8)E - 6 + (39 +/- 0.5).[GLU] and the correlation coefficient r = 0.9998. The limit of quantitation is 1.5 microM GLU and the relative standard deviation of the method 1.2%. The kinetics of the interfering TNBS hydrolysis reaction in alkaline range of pH, as well as the effect of sulfite concentration on the main reaction, are also presented. The analytical application of the reaction for the kinetic spectrophotometric assay of GLU and other amino acids, as well as TNBS, is presented and the relevant advantages and disadvantages of the method are discussed.     

Photoinactivation of hepatitis A virus by synthetic porphyrins

Porphyrins are photosensitizers and may be applicable in situations where viral inactivation is required, as for in vitro inactivation of nonenveloped viruses in blood components or in other aqueous media. No study has examined the efficacy of porphyrin inactivation on human pathogens such as hepatitis A virus (HAV) in plasma or other liquids. Experiments were conducted to evaluate the effect of synthetic porphyrins on HAV in porphyrin-containing human plasma and phosphate-buffered saline exposed to long-wavelength (365 nm) UV light. Inactivation of bacteriophage MS2 (MS2) also was determined in some trials. Solutions containing cationic, anionic or amphiphilic porphyrins irradiated with an average light dose of 4.3 J/cm(2) for 90 min resulted in >3 log(10) (>99.9%) to >4 log(10) (>99.99%) inactivation of both HAV and MS2. Viral inactivation may have been greater than observed because the limits of detection of the assay had been reached. Under ambient lighting conditions, none of the porphyrins was mutagenic in the Ames assay and only the congener with the longest chain-length, tetrakis (N-[n-hexadecyl]-4-pyridiniumyl) porphyrin, was appreciably toxic to mammalian cells. Disinfection by photoactivated synthetic porphyrins therefore can offer an effective and relatively safe approach to removal of nonenveloped viruses from aqueous media.     

Immobilization/stabilization of lipase fromCandida rugosa

With the aim of fixing the enzyme to the matrix by multiple covalent linkages, lipase from Candida rugosa (formerly cylindracea) has been insolubilized through its amino groups on Sepharose 6B previously activated with 2,3-epoxy-1-propanol. Two main variables that are known to control the number of bonds formed have been tested: the contact time between enzyme and activated support, and the temperature at which the immobilization reaction is carried out. Studies on activity and stability of the different derivatives prepared showed that higher temperatures and longer contact times lead to insolubilized enzymes that are more resistant to inactivation by temperature and the presence of organic solvents. At 50 degrees C and pH 7.2, the insoluble lipase was found to be 140 times more stable than its soluble counterpart.