Acid hydrolysis of protein in a microcapillary tube for the recovery of tryptophan

The acid hydrolysis of proteins was miniaturized and simplified by employing microcapillary tubes (100 microl in volume) with 6 M HCl containing 1% 2-mercaptoethanol and 3% phenol for an amino acid compositional analysis. The method not only eliminated the laborious evacuation step for the hydrolysis tube but also decreased the destruction of tryptophan during hydrolysis. The recovery of tryptophan was 79% by acid hydrolysis at 145 degrees C for 4 h. Since the acid mixture could be removed under vacuum, the hydrolysate was subjected to an amino acid analysis without neutralization or dilution.     

Partial vapor-phase hydrolysis of peptide bonds: A method for mass spectrometric determination of O-glycosylated sites in glycopeptides

In this study we present a method for determination of O-glycosylation sites in glycopeptides, based on partial vapor-phase acid hydrolysis in combination with mass spectrometric analysis. Pentafluoropropionic acid and hydrochloric acid were used for the hydrolysis of glycosylated peptides. The reaction conditions were optimized for efficient polypeptide backbone cleavages with minimal cleavage of glycosidic bonds. The glycosylated residues were identified by mass spectrometric analysis of the hydrolytic cleavage products. Although glycosidic bonds are partially cleaved under acid hydrolysis, the resulting mass spectra allowed unambiguous determination of the glycosylation sites. Examples are shown with mannosyl- and mucin-type glycopeptides. Performing the hydrolysis in vapor eliminates the risk for contamination of the sample with impurities from the reagents, thus allowing analysis of the reaction products without further purification both by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry.     

Carbohydrate analysis of water-soluble uronic acid-containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods.

Sulfuric acid hydrolysis according to the Saeman procedure, TFA hydrolysis, and methanolysis combined with TFA hydrolysis were compared for the hydrolysis of water-soluble uronic acid-containing polysaccharides originating from fungi, plants, and animals. The constituent sugar residues released were subsequently analyzed by either conventional GLC analysis of alditol acetates or high-performance anion-exchange chromatography with pulsed-amperometric detection. It was shown that TFA hydrolysis alone is not sufficient for complete hydrolysis. Sulfuric acid hydrolysis of these polysaccharides resulted in low recoveries of 6-deoxy-sugar residues. Best results were obtained by methanolysis combined with TFA hydrolysis. Methanolysis with 2 M HCl prior to TFA hydrolysis resulted in complete liberation of monosaccharides from pectic material and from most fungal and animal polysaccharides tested. Any incomplete hydrolysis could be assessed easily by HPAEC, by the detection of characteristic oligomeric products, which is difficult using alternative methods currently in use. Methanolysis followed by TFA hydrolysis of 20 micrograms water-soluble uronic acid containing polysaccharides and subsequent analysis of the liberated sugar residues by HPAEC allowed us to determine the carbohydrate composition of these polysaccharides rapidly and accurately in one assay without the need for derivatization.     

The evaluation of the temperature effect and hydrolysis conditions for amino acid analysis

A systematic investigation of the optimal temperature and hydrolysis time for amino acid analysis has been carried out under various conditions. It is found that some simplification and increase in speed relative to the conventional protocol of employing vacuum-sealed tubes and 110 C/24-72 hour hydrolysis can be achieved without loss of accuracy and performance in amino acid analyses of proteins and peptides. The effects of hydrolysis temperature and heating time on the recoveries of various labile and hydrophobic amino acids are exemplified in the hydrolysis of oxidized ribonuclease A, lysozyme and lens crystallin. The method provides a rapid processing of multiple samples within hours instead of days with the potential for the total automation of amino acid analysis starting from the preparation of protein hydrolysates.     

Carbohydrate composition analysis of bacterial polysaccharides: optimized acid hydrolysis conditions for HPAEC-PAD analysis.

The capsular polysaccharide from Haemophilus influenzae type b (polyribosyl ribitol-phosphate; PRP) and the capsular polysaccharides from Streptococcus pneumoniae types 6B, 14, 18C, and 23F (Pn6B, Pn14, Pn18C, and Pn23F) were subjected to acid hydrolysis using hydrofluoric (HF) and/or trifluoroacetic acid (TFA) and high-pH anion-exchange chromatography with pulsed amperometric detection in an effort to identify optimum hydrolysis conditions for composition analysis of their carbohydrate components. With the exception of PRP, composition analyses of polysaccharides containing a phosphate moiety in the repeating unit structure (Pn6B, Pn18C, and Pn23F) are significantly improved by subjecting the sample to HF hydrolysis (65 degrees C, 1 h) followed by TFA hydrolysis (98 degrees C, 16 h). This results in essentially quantitative hydrolysis of the phosphodiester bond to the carbohydrate components, which otherwise remained predominantly phosphorylated and poorly accounted for in the analysis. Optimum analysis of PRP was achieved following a 2-h hydrolysis with TFA at 80 degrees C, whereas Pn14 showed optimum results after a 16-h hydrolysis with TFA at 98 degrees C. These analyses also provide information about the relative susceptibility to acid hydrolysis of the various glycosidic and phosphodiester bonds in these polysaccharides, with evidence to suggest that the acid lability of a given bond can be dramatically different from one polysaccharide to another.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

Further study on gas phase acid hydrolysis of protein: improvement of recoveries for tryptophan, tyrosine, and methionine

A novel method for vapor phase acid hydrolysis of protein suitable for quantitative analysis of tryptophan is presented. The hydrolysis is carried out in vapor of a mixture made of 7 M HCl, 10% trifluoroacetic acid, and 20% thioglycolic acid in the presence of indole. Reasonably good recoveries of common amino acids, including tryptophan (above 75%), were achieved.     

A new approach for sample preparation of protein hydrolyzates for amino acid analysis

Sample preparations of protein hydrolyzates for amino acid analysis by ion-exchange chromatography has been accomplished without the removal of hydrochloric acid which was used for the hydrolysis. The technique involves partial neutralization of the available hydrochloric acid after hydrolysis with a solution which neutralizes and dilutes the sample hydrolyzate at the same time. The resulting sample solution which is employed for amino acid analysis produces an amino acid chromatogram having the same elution times and resolution as compared to a mixture of amino acids prepared in pH 2.2 sodium citrate buffer. Experimental data is also presented which shows that the amount of available hydrochloric acid in the final sample solution employed for amino acid analysis can affect both the resolution and elution time of many of the amino acids found in a protein hydrolyzate.     

Kinetic modeling analysis of maleic acid-catalyzed hemicellulose hydrolysis in corn stover

Maleic acid-catalyzed hemicellulose hydrolysis reaction in corn stover was analyzed by kinetic modeling. Kinetic constants for Saeman and biphasic hydrolysis models were analyzed by an Arrhenius-type expansion which include activation energy and catalyst concentration factors. The activation energy for hemicellulose hydrolysis by maleic acid was determined to be 83.3 +/- 10.3 kJ/mol, which is significantly lower than the reported E(a) values for sulfuric acid catalyzed hemicellulose hydrolysis reaction. Model analysis suggest that increasing maleic acid concentrations from 0.05 to 0.2 M facilitate improvement in xylose yields from 40% to 85%, while the extent of improvement flattens to near-quantitative by increasing catalyst loading from 0.2 to 1 M. The model was confirmed for the hydrolysis of corn stover at 1 M maleic acid concentrations at 150 degrees C, resulting in a xylose yield of 96% of theoretical. The refined Saeman model was used to evaluate the optimal condition for monomeric xylose yield in the maleic acid-catalyzed reaction: low temperature reaction conditions were suggested, however, experimental results indicated that bi-phasic behavior dominated at low temperatures, which may be due to the insufficient removal of acetyl groups. A combination of experimental data and model analysis suggests that around 80-90% xylose yields can be achieved at reaction temperatures between 100 and 150 degrees C with 0.2 M maleic acid.     

Effect of sulfate groups from sulfuric acid hydrolysis on the thermal degradation behavior of bacterial cellulose

When used as fillers in polymer composites, the thermostability of cellulose crystals is important. Sulfate groups, introduced during hydrolysis with sulfuric acid, are suspected to diminish the thermostability. To elucidate the relationship between the hydrolysis conditions, the number of sulfate groups introduced, and the thermal degradation behavior of cellulose crystals, bacterial cellulose was hydrolyzed with sulfuric acid under different hydrolysis conditions. The number of sulfate groups in the crystals was determined by potentiometric titration. The thermal degradation behavior was investigated by thermogravimetric analysis. The sulfate group content increased with acid concentration, acid-to-cellulose ratio, and hydrolysis time. Even at low levels, the sulfate groups caused a significant decrease in degradation temperatures and an increase in char fraction confirming that the sulfate groups act as flame retardants. Profile analysis of the derivative thermogravimetric curves indicated thermal separation of the degradation reactions by the sulfate groups into low- and high-temperature processes. The Broido method was used to determine activation energies for the degradation processes. The activation energies were lower at larger amounts of sulfate groups suggesting a catalytic effect on the degradation reactions. For high thermostability in the crystals, low acid concentrations, small acid-to-cellulose ratios, and short hydrolysis times should be used.     

Determination of dopamine and methoxycatecholamines in patient urine by liquid chromatography with electrochemical detection and by capillary electrophoresis coupled with spectrophotometry and mass spectrometry

The applicability of capillary electrophoresis (CE) with UV and mass spectrometric (MS) detection for the determination of dopamine and methoxycatecholamines in urine was evaluated in comparison with the liquid chromatography-electrochemical detection (LC-EC) method widely used in catecholamine analysis. The catecholamines in urine were deconjugated with acid or enzyme hydrolysis, purified by cation exchange (CEX) or solid-phase extraction (SPE) with a copolymer of N-divinylpyrrolidone and divinylbenzene and analyzed by LC-EC, CE-UV, and CE-MS. Acid hydrolysis was more effective in the deconjugation than enzymatic hydrolysis with Helix pomatia. However, the recoveries of HMBA, DA and NMN from spiked samples were less than 30% after acid hydrolysis and SPE purification. The CEX purification was more efficient than SPE in removing matrix compounds from the urine samples. The limits of detection were lower in LC-EC analysis than in CE-UV or CE-MS. Many factors in the analytical procedure caused deviations in the concentrations measured for urinary dopamine and methoxycatecholamines. The recovery of HMBA, which was used as the internal standard, was poor after acid hydrolysis and SPE purification. The purification methods were validated in conjunction with the analytical methods and therefore cross analysis was unsuccessful. The LC-EC method was the most sensitive, but CE-UV and CE-MS were sensitive enough for the determination of dopamine and methoxycatecholamines even in healthy patient urine. The EC and MS detections were superior to the UV detection in specificity since, after acid hydrolysis, some matrix compounds were migrating close to I.S., DA and 3MT.     

Evaluation of immunocytochemical detection methods of incorporated bromodeoxyuridine in hybridomas

Bivariate distributions obtained from nominal acid hydrolysis or thermal treatment methods used in the cell cycle analysis of incorporated bromodeoxyuridine were shown to be unacceptable with hybridomas. Four different cell treatment and staining methods were compared. These methods are acid hydrolysis, thermal denaturation, nuclei extraction with pepsin digestion, and simultaneous pepsin digestion and acid hydrolysis. The nuclei extraction method was determined to be the most appropriate for the immunocytochemical staining of incorporated bromodeoxyuridine in hybridomas. The resulting bivariate distribution provides a clear distinction between labelled and unlabelled cell fractions. The method based on nuclei extraction with pepsin digestion was optimized for a hybridoma line used in this study.     

Analysis for amide nitrogen in proteins

Existing procedures for amide analysis of soluble proteins have been studied. Use of the automatic amino acid analyser for the determination of released ammonia gave greatly increased sensitivity over the distillation procedure and more reliable blank values. For the insoluble fibrous proteins, wool and silk hydrolysis in vacuo was found to remove the need for an extrapolation procedure; prolonged hydrolysis was required with hydrochloric acid but not with hydriodic acid.     

Identification of iodotyrosines and some iodothyronines as dimethylaminoazobenzenethiohydantoin derivatives. Application to the sequencing of iodoamino acid containing peptides

A method has been developed for the gas chromatographic analysis of lipoic acid in biological samples. The lipoic acid is released from the samples by acid hydrolysis in the presence of the internal standards 1,2-dithiolane-3-butyric acid and/or 1,2-dithiolane-3-caproic acid. After hydrolysis, the lipoic acid and the internal standards are extracted from the hydrolysate and converted into the S,S-dibenzylmethyl esters. Gas chromatographic analysis of this mixture completely separates each of the homolog derivatives from the lipoic acid derivative and allows for the quantitation of the lipoic acid in the sample. Samples containing more than ～50 ng of lipoic acid can be easily assayed. Results are presented that show that the lipoic acid content of Escherichia coli depends on the carbon source used for its growth.     

A new chemical tool for characterization and partial depolymerization of red algal galactans

Complete acid hydrolysis of red algal galactans in the presence of borane - 4-methylmorpholine complex has been shown to prevent the acid degradation of 3,6-anhydrogalactose derivatives by their reduction to the corresponding 3,6-anhydro-galactitols, whereas all the other monosaccharides are liberated essentially in the non-reduced form; the reductive hydrolysis products may be determined quantitatively using gas-liquid chromatography (GLC). The method is recommended for preliminary characterization of the polysaccharide composition of red algal biomass. Partial acid hydrolysis of galactans in the presence of the same reducing agent gives rise to reduced oligosaccharides having terminal 3,6-anhydrogalactitol residues. Based on this reaction, the attribution of unknown galactans to the agar or carrageenan groups is possible by partial reductive hydrolysis of small samples of algal biomass with subsequent identification of agarobiitol or carrabiitol acetates by GLC. Sulfate groups are substantially retained under partial reductive hydrolysis conditions; the isolation by liquid chromatography and elucidation of structures of reduced sulfated oligosaccharides may be of great value for the structural analysis of complex red algal galactans.     

A method for chromatographic separation and fluorometric quantification of 2'',3''-cyclic nucleotide-3''-phosphodiesterase reaction products

Treatment of hydrochloric acid with sodium sulfite prior to the acid hydrolysis of bovine pancreatic ribonuclease A has been found to suppress the oxidation of cystine, methionine, and tyrosine without adversely affecting the recoveries of other amino acids. Statistical analysis of the results indicated that the assumption of the independence of the mean and the variance, an assumption commonly used in the evaluation of the effects of various treatments, may not be valid in evaluating antioxidants used in the acid hydrolysis of proteins.     

Structural studies on ascophyllan and the fucose-containing complexes from the brown alga Ascophyllum nodosum

The two major “complexes” isolated from a dilute-acid extract of Ascophyllum nodosum were degraded by mild, acid hydrolysis to a fucan portion and an ascophyllan-like portion. Protein was enriched in the ascophyllan-like fraction. A time-course study of the acid hydrolysis, together with data on digestion by pronase and hydrolysis by base indicated that the complexes were formed by a fucan backbone to which varyious numbers of ascophyllan-like molecules were attached by an acid-labile linkage. The primary uronic acid in the ascophyllan-like portion of the complex was mannuronic acid, whereas the free ascophyllan isolated in this study contained a mixture of guluronic, mannuronic, and glucuronic acids*. In each of these uronic acid-rich materials, hydrolysis by base suggested a uronic acid backbone having relatively long, fucose-containing, side chains. Hydroxyproline was present in the complex, but did not appear to be part of the major carbohydrate-protein linkages. A protein-enriched fraction was obtained by treatment of the ascophyllan-like portion of the complex with oxalic acid. Amino acid analysis, before and after treatment with mild base suggested that serine and threonine were involved in the linkage of protein to carbohydrate.     

Plant protein hydrolysates as fish fry feed in aquaculture. Hydrolysis of rapeseed proteins by an enzyme complex from king crab hepatopancreas

Rapeseed proteins were processed by an enzyme complex isolated from king crab hepatopancreas in order to obtain a hydrolysate for use as fish fry feed. The amino acid composition of the obtained protein preparation was close to the amino acid composition of fishmeal traditionally used in the production of fish feed. SDS-PAGE, HPLC, and mass spectrometric analysis of the products of enzymatic hydrolysis of rapeseed proteins showed that the proteins were hydrolyzed to a high degree. The composition of the hydrolysates depended on the hydrolysis time and included free amino acids (27% of the total weight of the protein mix after 3 h of hydrolysis and 56% after 21 h of hydrolysis), short peptides (2 to 20 amino acid residues), and small amounts of protein fragments with a molecular weight of approximately 14 kDa, as shown by by SDS-PAG electrophoresis.     

Gas chromatographic–mass spectrometric analysis of hydroxylamine for monitoring the metabolic hydrolysis of metalloprotease inhibitors in rat and human liver microsomes

A gas chromatographic–mass spectrometric (GC–MS) method was developed for the analysis of hydroxylamine (HA) in supernatants obtained from liver microsomes. HA monitoring was used to determine the metabolic hydrolysis of two hydroxamic acid-based matrix metalloprotease inhibitors in rat and human liver microsomes. The hydrolysis of the hydroxamic acids to their corresponding carboxylic acids releases HA as a common metabolic product. HA was derivatized to acetone oxime by addition of acetone to the liver microsomal supernatant, followed by direct injection of the supernatant into the GC–MS, with detection of the oxime by selected-ion-monitoring. The method is simple, reproducible, and sensitive for the determination of the hydrolysis of hydroxamic acid compounds, where hydrolysis is the major metabolic pathway. The methodology can be used for rank ordering and selecting hydroxamic acid analogs based on their susceptibility to hydrolysis.     

Studies on the Chemical Structure of Konjac Mannan

Rate-constants of β-d-mannosidic and β-d-glucosidic linkages for acid hydrolysis were determined. And from these results, statistical analysis of acid hydrolysis of the main chain of the mannan was made whereby the yields of the fragmental disaccharicles could be calculated for any degree of hydrolysis. The symmetry of the result of these calculations with those observed experimentally may support the proposed chemical structure of the mannan. Moreoyer, from the result of action of cellulase on the manno-oligosaccharides, the main process of the reaction, “hydrolysis of the mannan by cellulase” was discussed.