Determination of methylated basic, 5-hydroxylysine, elastin crosslinks, other amino acids, and the amino sugars in proteins and tissues

Analytical single-column chromatographic methods have been developed for determining all methylated basic amino acids, isodesmosine, desmosine, the amino sugars glucosamine and galactosamine, the diastereoisomers of 5-hydroxylysine, and related compounds at picomole levels in protein and tissue hydrolysates. Complete resolution of all these unique basic amino acids as discrete peaks was achieved in 5.4 on a 50 X 0.28-cm microcolumn of Dionex type DC-4A spherical resin (9.0 +/- 0.5 micron) using updated instrumentation commonly available for amino acid analysis. The column was operated at 5.65 ml/h with two 0.35 M sodium citrate buffers (pH 5.700 and 4.501), at two temperatures (31.5 and 73 degrees C). Excellent resolution of all omega-N-methylarginines and related compounds was also achieved in 3 h using a 17.5 X 0.28-cm microcolumn of Dionex DC-5A resin (sized to 6.0 +/- 0.5 microns), two citrate buffers (0.21 M Na+, pH 5.125; 0.35 M Na+, pH 5.700), a buffer flow rate of 5.75 ml/h, and a temperature of 52 degrees C. Complete separation of all other amino acids found in protein or tissue hydrolysates including S-carboxymethyl cysteine, 4-hydroxyproline, methionine S,S-dioxide, and the amino sugars was also carried out in 95 min using a 23.5 X 0.28-cm microcolumn of Dionex DC-5A resin. The use of purified microcolumn buffers gave smooth baselines without interference from artifacts or minor hydrolysate components. The major advantages of these methods are: first, their high resolving power; second, their high sensitivity which is comparable and in some aspects superior to the newer instruments; and third, their high reproducibility (100 +/- 2.5%) and low operating costs. These methods should be especially valuable for determining myosin, actin, and elastin in tissue hydrolysates from the amounts of N tau-methylhistidine, desmosine, or isodesmosine present, respectively, and for studying protein methylation, hydroxylation, cross-linking formation, and the turnover rates of contractile and connective tissue proteins in biological systems.     

Single-column amino acid analysis of connective tissue proteins and related materials.

A single-column procedure is described which, in general, is useful for the amino acid analysis of any simple or complex protein, but in particular the procedure is useful for the separation and quantitation of the amino acids and amino sugars, if present, in collagen, elastin and related materials from a variety of connective tissues. In addition to the amino acids commonly found in proteins, the system resolves hydroxyproline, hydroxylysine, desmosine, isodesmosine, glucosamine, galactosamine and also a number of other ninhydrin-positive compounds ordinarily not found in acid hydrolysates of proteins. Chromatograms of the analysis of a synthetic mixture of amino acid standards and also collagen and elastin hydrolysates indicate the multiple use of the procedure and the nearbaseline separation of all the amino acids. The basic amino acids are well spread, thus providing flexibility for the isolation and identification of additional ninhydrin-positive components. The analysis, which requires approximately 2 hr and four sodium citrate buffers was performed with a Durrum (D-500) amino acid analyzer.     

Separation of o-phthalaldehyde derivatives of amino acids of the internal pool of yeast by reverse-phase liquid chromatography

Summary Derivatization of primary amino acids with orthophthalaldehyde and -mercaptoethanol forms derivatives that can be detected by absorbance at 340 nm. These were separated by reverse-phase high performance liquid chromatography (HPLC). A step- or more complex gradient (acetonitrile/phosphate buffer gradient) was used. The effects of several parameters (pH, ionic strength, etc) were characterized and used to design a rapid separation of the amino acids commonly found in physiological fluids. The method described is rapid, sensitive and precise as sensitivity limits are about 25 pmol and the separation time, injection to injection, is 16 min.     

Development of an improved automated gas-chromatographic chiral analysis system: application to non-natural amino acids and natural protein hydrolysates

In the use of combinatorial chemistry as a novel strategy for drug discovery, the chirality assessment of building blocks used for library construction is particularly important in the evaluation of biological actions of generated libraries. The procedure for chiral analysis of lead compounds in screening may be of a high priority, particularly in the case of the protection of intellectual rights. Previously, an automated amino acid analysis system using enantiomer labeling was developed. The system incorporates a reactor, which allows automated esterification and acylation of amino acids, and is connected to an on-line gas chromatographic system. A capillary column coated with a chiral phase is employed for the separation of the enantiomers. This particular system is improved with a newly constructed "high-throughput auto-derivatizer" in combination with a new GC-system. The resulting data can be processed by newly constructed software. The analyses of amino acid derivatives or hydrolysates of proteins and peptides are carried out routinely within ca. 45 min, including derivatization. Using this system several non-natural amino acids were tested with respect to the stereoisomeric configuration. In addition, acid hydrolysates of food proteins and tissues obtained by autopsy were analyzed as an application to proteome research.     

Amino acid analysis by capillary electrophoresis after phenylthiocarbamylation

Capillary electrophoresis using SDS in phosphate buffer provides high resolution and short separation time for peptide and protein hydrolysate amino acids after derivatization with phenylisothiocyanate. The phenylthiocarbamyl derivatives are quantified in the picomole and femtomole range at signal-to-noise ratios better than 3:1 (for 50 fmol) and with a linearity correlation coefficient averaging 0.9938. The migration time and peak area variabilities were on average 1.1 and 2.7%, respectively. Complete separation of all the 18 amino acids normally found in polypeptide hydrolysates is achieved in less than 30 min using 75-microm capillaries while 50-microm capillaries require less than 15 min. Analysis of peptide and protein hydrolysates in the range 10-600 residues revealed excellent agreement with the known compositions at sensitivities better by large factors than the corresponding HPLC methodology (about 20-fold) and conventional ninhydrin-based analysis (about 1000-fold).     

Utilisation of Chlorella vulgaris cell biomass for the production of enzymatic protein hydrolysates

Studies on enzymatic hydrolysis of cell proteins in green microalgae Chlorella vulgaris 87/1 are described. Different proteases can be used for production of hydrolysates from ethanol extracted algae. The influence of reaction parameters on hydrolysis of extracted biomass with pancreatin was considered, and the composition of hydrolysates (Cv-PH) was investigated in relation to the starting materials. Significant changes in the degree of hydrolysis were observed only during the first 2h and it remained constant throughout the process. An enzyme-substrate ratio of 30-45 units/g algae, an algae concentration of 10-15% and pH values of 7.5-8.0 could be recommended. Differences in the chromatographic patterns of Cv-PH and a hot-extract from Chlorella biomass were observed. Adequate amounts of essential amino acids (44.7%) in relation to the reference pattern of FAO for human nutrition were found, except for sulfur amino acids. Cv-PH could be considered as a potential ingredient in the food industry.     

Optimum conditions of autoclaving for hydrolysis of proteins and urinary peptides of prolyl and hydroxyprolyl residues and HPLC analysis

A method for urinary peptide(s) and protein hydrolysis, involving autoclaving at 15psi (121 degrees C) for 60min, is described. Using three candidate proteins (bovine serum albumin, casein and gelatin) and urine specimens, the effect of autoclaving with respect to the optimum time required for hydrolysis under both acidic (6N HCl) and alkaline (6N KOH) conditions was studied. Recoveries of total amino acids from proteins and urine hydrolysate(s) suggest that complete hydrolysis of proteins and urinary peptides could be achieved by autoclaving for 30-60min instead of 16h of incubation at 110 degrees C. Further, stability of some of the individual amino acids was also studied. The observed differential stability of amino acids under acidic and alkaline conditions, as demonstrated in this study by HPLC analysis, makes it imperative to choose the appropriate hydrolytic condition while studying the composition of any given amino acids in urinary peptide(s)/protein hydrolysates. Further, the finding that both Pro and Hyp were stable under alkaline conditions of hydrolysis by autoclaving renders this method suitable for assaying these two amino acids from urine hydrolysates, hence its utility in the study of urinary peptide derived Hyp and Pro in bone/cartilage disorders.     

Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.     

Amino acid and soluble protein cocktail from waste keratin hydrolysed by a fungal keratinase of Paecilomyces marquandii

Waste bovine hooves and horns were enzymatically hydrolysed into soluble products intended for foliar fertilizer. With the powdered keratin at 50°C and pH 8 between 34 to nearly 60% of nitrogen was solubilized in 5 h, depending on the enzyme concentration. The reaction could further be improved by steam pretreatment of the keratin, resulting in 98% solubilisation of the nitrogen. The products of hydrolysis consisted of a mixture of soluble proteins, peptides, and free amino acids. Among the latter, 18 common amino acids were detected. Several of them were previously recognized to have a positive effect on plants. Nonpolar neutral, basic, and sulphur amino acids were present in relatively large amounts, while proline and tryptophan were not found. Comparison with other protein hydrolysates aimed for fertilizer suggests that keratin degradation products, obtained by enzymatic hydrolysis, have potential to be used for foliar fertilization, alone or in a combination with another complementary hydrolysate of a different source, such as skin or plant proteins.     

Routine amino acid capillary column chromatography of microquantities in the picomole range--procedures and modifications of a commercial analyzer

A commercial micro amino acid analyzer using capillary columns (internal diameter 0.7 mm) had to be modified greatly in order to get satisfactory separation and quantitative analysis on the microscale of free amino acids in biological fluids, tissue extracts, and hydrolysates. Furthermore, the usual analytical conditions were changed (i.e., lowering of the buffer pH) and a procedure and apparatus for sample deproteinization is described which allows simple handling of volumes in the range from 20 to 100 μl. The modifications described guarantee better and more reproducible analyses than reported so far. A design of a new “analytical unit” for capillary column chromatography is proposed.     

Single column analysis of amino acids in protein hydrolysates utilizing the fluorescamine reaction

A system is described for the separation of the amino acids commonly found in protein hydrolysates at the picomole level using a single ion exchange column and for their quantitation by the fluorescamine (4-phenylspiro[furan-2 (3H),1′-phthalan]-3,3′-dione) reaction. Three sodium citrate buffers were required for the separation of the amino acids with an analysis time of approximately 3 hr. The amino acids in 1 μg of hydrolyzed bovine serum albumin were separated using a single ion exchange column and were detected in the effluent from the column by the fluorescamine assay. The results were compared with those obtained using a commercial amino acid analyzer and 150 μg of hydrolyzed bovine serum albumin. The chromatogram produced by the more sensitive analyzer utilizing the fluorescamine reaction to detect the amino acids compared favorably with the chromatogram produced by the commercial analyzer utilizing the ninhydrin reaction with the exception that the proline peak was missing. Proline and hydroxyproline fail to yield fluorescence on reaction with fluorescamine unless converted from imines to primary amines.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based amino acid analysis

Amino acid analysis has been an integral part of analytical biochemistry for more than 50 years. However, its experimental design, which includes derivatization of amino acids followed by some kind of chromatographic separation, has not changed over the years. We have developed a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-based method for the quantitative analysis of amino acids. This method does not require any amino acid modification, derivatization, or chromatographic separation. The data acquisition time is decreased to several seconds for a single sample. No significant ion suppression effects were observed with the developed sample deposition technique, and the method was found to be reproducible. Linear responses between the amino acid concentration and the peak intensities ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 microM, and correlation coefficients were between 0.983 (for arginine) and 0.999 (for phenylalanine). Limits of quantitation were between 0.03 microM (for arginine) and 3.7 microM (for histidine and homocysteine). This method was applicable to the mixtures of free amino acids as well as to HCl hydrolysates of proteins. Furthermore, we have shown that this method can be applied to other biologically important low-molecular weight compounds such as glucose.     

Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level

A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

A trial to determine D-amino acids in tissue proteins of mice

Summary Eleven neutral amino acids and two acidic amino acids in tissue proteins of mouse kidney, liver and brain were analyzed for the presence of D-enantiomers. The proteins were hydrolyzed with HCl for 6 h. Of the thirteen amino acids investigated, the presence of D-enantiomers of serine, alanine, proline, aspartate and glutamate (including asparagine and glutamine) was shown in the hydrolysates. However, the level of D-enantiomers were not significantly higher than that of 6-h hydrolysate of serum albumin examined as a control protein. Serum albumin was shown to contain no D-amino acid residues.     

An improved method for the determination of γ-carboxyglutamic acid in proteins,bone, and urine

A method of high-performance liquid chromatographic separation of the fluorescence derivative of γ-carboxyglutamic acid (Gla) is presented. Alkaline hydrolysates of protein samples were reacted with o-phthalaldehyde in the presence of ethanethiol for 2 min, and the fluorescence derivative of γ-carboxyglutamic acid was resolved from the other amino acids by a short column packed with silica-based anion exchanger under isocratic conditions. By this method, as low as 200 fmol of γ-carboxyglutamic acid can be quantitatively analyzed within 10 min. The method presented here shortened the analysis time for Gla and was at least 10 times more sensitive than the method we described previously (Anal. Biochem.117, 259–265, 1981). The application of this method to the formic acid-soluble or insoluble γ-carboxyglutamic acid-containing proteins in chicken bone and the concomitant increase of γ-carboxyglutamic acid content in chicken bone with age are reported.     

Determination of radioactivity of proline and hydroxyproline in tissue hydrolysates after the elimination of interference by primary amino acids by deamination

A simple method for the determination of radioactivity of proline and hydroxyproline, particularly of small amounts, in hydrolysates of tissues is described. Specificity is assured by eliminating primary amino acids from the hydrolysates by deamination and then extraction before separation of proline from hydroxyproline by paper chromatography. Six to eight tissue samples may be compared simultaneously. The efficiency and reproducibility are good, as indicated by the use of labeled l-proline, labeled dl-hydroxyproline, a hydrolysate of a protein in which the amino acids (and proline) were labeled, and hydrolysates of tissues cultured in media containing radioactive l-proline. The method is particularly useful when ion-exchange column chromatography of amino acids is not in routine use.     

Phosphate Acceptor Amino Acid Residues in Structural Proteins of Rhabdoviruses

Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses.     

Optimization of enzymatic hydrolysis of visceral waste proteins of Catla (Catla catla) for preparing protein hydrolysate using a commercial protease

Protein hydrolysate was prepared from visceral waste proteins of Catla (Catla catla), an Indian freshwater major carp. Hydrolysis conditions (viz., time, temperature, pH and enzyme to substrate level) for preparing protein hydrolysates from the fish visceral waste proteins were optimized by response surface methodology (RSM) using a factorial design. Model equation was proposed with regard to the effect of time, temperature, pH and enzyme to substrate level. An enzyme to substrate level of 1.5% (v/w), pH 8.5, temperature of 50 degrees C and a hydrolysis time of 135 min were found to be the optimum conditions to obtain a higher degree of hydrolysis close to 50% using alcalase. The amino acid composition of the protein hydrolysate prepared using the optimized conditions revealed that the protein hydrolysate was similar to FAO/WHO reference protein. The chemical scores computed indicated methionine to be the most limiting amino acid. The protein hydrolysate can well be used to meet the amino acid requirements of juvenile common carp and hence has the potential for application as an ingredient in balanced fish diets.     

Identification of products of protein phosphorylation in T37-transformed cells and comparison with normal cells

Proteins from crown gall tissue labelled in vivo with [³²P]orthophosphate were analysed by SDS-polyacrylamide gel electrophoresis. The major phosphorylated proteins were of 50.6 and 48.3 kDa, with minor bands at 80.1, 73.9, 68, 40.4, 30, 21.5, 20.2 and 15.2 kDa. Partial hydrolysates of total ³²P-labelled proteins were analysed in a number of ways. A two-dimensional separation on paper by electrophoresis in pyridine/acetic acid at pH 3.5 followed by chromatography in isobutyric acid/0.5 M ammonia revealed radioactive spots coincident with phosphoserine and phosphothreonine markers and only partially coincident with the phosphotyrosine marker. Two-dimensional electrophoresis at pH 1.9 followed by pH 3.5, however, unequivocally showed the presence of phosphotyrosine after elution of the phosphotyrosine marker. Phosphoserine, phosphothreonine and phosphotyrosine were present in the ratio 89.4:8.5:2.1. This is a much higher level of phosphotyrosine than normally found in animal cells. The three phosphoamino acids were confirmed by chromatography with authentic samples in four solvent systems on cellulose or silica TLC, and by dansylation followed by silica TLC. The radioactive compound running almost coincident with phosphotyrosine on two-way electrophoresis, pH 3.5, followed by chromatography in isobutyric acid/0.5 M ammonia was identified tentatively as uridine 5′-monophosphate on the basis of electrophoretic and chromatographic behaviour. Further experiments to compare normal (growing and non-growing) tobacco callus and T37-transformed cells did not give markedly different ratios of the three phosphoamino acids, although the rapidly-growing normal tobacco (i.e., plus cytokinin) appeared to have a greater abundance of the two minor phosphoamino acids (approx. 2-times). The lack of effect of transformation is in contrast to animal cells where transformation results in a 10-fold increase in the virally affected cells.     

A non-specific aminopeptidase from Aspergillus

A fermentation broth supernatant of the Aspergillus oryzae strain ATCC20386 contains aminopeptidase activity that releases a wide variety of amino acids from natural peptides. The supernatant was fractionated by anion exchange chromatography. Based on the primary amino acid sequence data obtained from proteins in certain fractions, polymerase chain reaction (PCR) primers were made and a PCR product was generated. This PCR product was used to screen an A. oryzae cDNA library from which the full length gene was then obtained. Fusarium venenatum and A. oryzae were used as hosts for gene expression. Transformed strains of both F. venenatum and A. oryzae over-expressed an active aminopeptidase (E.C. 3.4.11), named aminopeptidase II. The recombinant enzyme from both fungal hosts appeared as smears on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After deglycosylation of the N-linked sugars, both samples were a sharp band at approximately 56 kDa and had identical N-terminal amino acid sequences. Aminopeptidase II is a metalloenzyme with, presumably, Zn in the active site. Using various natural peptides and para-nitroanilides (pNAs) of amino acids as substrates, the aminopeptidase was found to be non-specific. Only X-Pro bonds demonstrated resistance to hydrolysis catalyzed by this aminopeptidase. The optimal enzyme activity was observed at pH 9.5 and 55 degrees C. Among amino acid pNAs, Leu-pNA appears to have the highest value of bimolecular constant of 40 min(-1) mM(-1) (k(cat) = 230 min(-1); K(m) = 5.8 mM) at pH 7.5 and 21 degrees C. Among Xaa-Ala-Pro-Tyr-Lys-amide pentapeptides, the velocity of catalytic hydrolysis at pH 7.5 and 21 degrees C was in a decreasing order: Pro, Ala, Leu, Gly and Glu.