Microwave assisted facile synthesis of amino acid benzyl ester p-toluenesulfonate and hydrochloride salts

A simple method for the synthesis of several amino acidbenzyl ester p-toluenesulfonate salts from thecorresponding amino acid and benzyl alcohol in presence of p-toluenesulfonic acid accelerated with microwave irradiation isdescribed. Under similar condition, the amino acid benzyl esterhydrochloride salts have also been obtained by using thionylchloride instead of p-toluenesulfonic acid in good yieldand purity.     

The synthesis of 1,2:5,6-di-O-isopropylidene-d-mannitol: a comparative study

Three different methods of acetonation of d-mannitol using (a) acetone and zinc chloride, (b), 2,2-dimethoxypropane, 1,2-dimethoxyethane, and tin(II) chloride, and (c) 2-methoxypropene, N,N-dimethylformamide, and p-toluenesulfonic acid were studied in detail and compared, using gas-liquid chromatographic techniques. In each reaction, isomeric diacetals are formed, but method a gives the 1,2:5,6-diacetal in the highest yield (63%). Methods b and c give a more complex mixture of acetals than proposed in the literature, and both methods are less economical than a. A new 1,2:3,6:4,5-tri-O-isopropylidene-d-mannitol could be separated, and its graded hydrolysis was compared to that of the 1,2:3,4:5,6-triacetal.     

Determination of tryptophan as the reduced derivative by acid hydrolysis and chromatography

A new procedure for the analyses of tryptophan and the total amino acid composition of proteins was based on the observations that pyridine borane reduces tryptophan in trifluoroacetic acid, while other amino acids remain intact [M. Kurata, Y. Kikugawa, T. Kuwae, I. Koyama, and T. Takagi (1980) Chem. Pharm. Bull. 28, 2274-2275; W.S.D. Wong, D.T. Osuga, and R.E. Feeney (1984) Anal. Biochem. 139, 58-67]. Concentrated HCl was used instead of trifluoroacetic acid for analytical purposes. The products were stable to hydrolysis in 6 N HCl, and the reduction did not interfere with hydrolysis and subsequent analyses. Quantitative recovery was achieved with most proteins when they were subjected to acid reduction in ice-cooled concentrated HCl with two incremental additions of pyridine borane. The reaction was terminated after 10 min by dilution with an equal volume of H2O, vacuum sealing, and hydrolyzing at 110 degrees C for 22 h. The yields of the expected values for cytochrome c, catalase, bovine serum albumin, subtilisin BPN', trypsin, chymotrypsin, beta-lactoglobulin, lysozyme, and pepsin were obtained. Ovotransferrin and ovalbumin, however, yielded values for tryptophan lower than literature values. With two different ion-exchange methods, the recoveries of all other amino acids were comparable to those obtained by acid hydrolysis with 6 N HCl. Since the same hydrolysate can be analyzed for both tryptophan and all the other amino acids, the procedure is a more convenient method than those requiring separate determinations. Initial results indicate that the method may be applied to high-performance liquid chromatographic procedures with adaptations of the protocols if necessary.     

Decomposition of phosphoserine and phosphothreonine during acid hydrolysis

The rate of decomposition of phosphoserine and phosphothreonine, both as free O-phosphoamino acids and in peptides, was studied under conditions of acid hydrolysis, using 1, 2, and 6 n HCl at 110°C. For the free O-phosphoamino acids, the decomposition follows first-order kinetics and is two- to fourfold faster for phosphoserine than phosphothreonine. The rate of destruction of these O-phosphoamino acids during hydrolysis of peptides is dependent on the neighboring amino acid residues, and thus the hydrolysis of a free O-phosphoamino acid generally is not a good model for the hydrolysis of that O-phosphoamino acid in a peptide. For the three peptides studied, maximal recoveries of O-phosphoamino acids are obtained after hydrolysis in 6 n HCl for 2 to 4 hr.     

The preparation of matrix-bound proteases and their use in the hydrolysis of proteins

Four proteases, crude acid protease from Aspergillus, pronase, amino-peptidase M, and prolidase, have been covalently attached to activated agarose and to amino propyl glass beads. The matrix-bound enzymes have been tested as catalysts for the complete hydrolysis of protein substrates, with the primary goal to isolate unstable amino acid derivatives present in the substrate protein. Under conditions used in the present work, the total amino acid release from the protease-catalyzed hydrolysis of four substrate proteins (pancreatic ribonuclease, egg white lysozyme, yeast enolase, and bovine insulin) was 95–103% of that observed in standard acid hydrolysis. Recovery of individual amino acids showed greater deviation from the theoretical values, but cystine was the only amino acid recovered in low yields (42–77%) from all four proteins. Derivatized amino acids, such as methionine sulfoxide, O-(butylcarbamoyl)-serine, and N-glycosyl asparagine have been obtained from chemically modified proteins or from unmodified glycoprotein in good yield, and normal amino acid constituents of proteins which cannot be quantified after acid hydrolysis (tryptophan, asparagine, and glutamine) have also been determined either directly after proteolysis or after proteolysis in conjunction with acid hydrolysis.     

Nonspecific cleavage of proteins using graphene oxide

In this article, we report the intrinsic catalytic activity of graphene oxide (GO) for the nonspecific cleavage of proteins. We used bovine serum albumin (BSA) and a recombinant esterase (rEstKp) from the cold-adapted bacterium Pseudomonas mandelii as test proteins. Cleavage of BSA and rEstKp was nonspecific regarding amino acid sequence, but it exhibited dependence on temperature, time, and the amount of GO. However, cleavage of the proteins did not result in complete hydrolysis into their constituent amino acids. GO also invoked hydrolysis of p-nitrophenyl esters at moderate temperatures lower than those required for peptide hydrolysis regardless of chain length of the fatty acyl esters. Based on the results, the functional groups of GO, including alcohols, phenols, and carboxylates, can be considered as crucial roles in the GO-mediated hydrolysis of peptides and esters via general acid–base catalysis. Our findings provide novel insights into the role of GO as a carbocatalyst with nonspecific endopeptidase activity in biochemical reactions.     

Microwave assisted facile synthesis of amino acid benzyl ester p-toluenesulfonate and hydrochloride salts

Summary A simple method for the synthesis of several amino acid benzyl esterp-toluenesulfonate salts from the corresponding amino acid and benzyl alcohol in presence ofp-toluenesulfonic acid accelerated with microwave irradiation is described. Under similar condition, the amino acid benzyl ester hydrochloride salts have also been obtained by using thionyl chloride instead ofp-toluenesulfonic acid in good yield and purity.     

A precise method for the quantitation of proteins taking into account their amino acid composition.

A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-μl solutions containing 1 to 100 μg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per aliquot of hydrolysate analyzed can be determined.     

Recovery of tryptophan from 25-minute acid hydrolysates of protein

It was found that thioglycolic acid prevents destruction of tryptophan during rapid hydrolysis of protein with a trifluoroacetic acid/HCl mixture (1:2, v/v) at 166 degrees C for 25 or 50 min. The addition of 5% (v/v) thioglycolic acid gave the maximum tryptophan recovery (88.3%) for a 25-min hydrolysate of lysozyme. Tryptophan recoveries varied slightly among three different proteins; 88% for lysozyme, 73% for alpha-chymotrypsinogen A, and 85% for apomyoglobin. However, when extrapolated to zero time, the values were close to one another: 94, 87, and 88%, respectively. The addition of thioglycolic acid was also advantageous for recovering amino acids other than tryptophan. Particularly, yields of carboxymethylcysteine and methionine were greatly improved. This modified rapid hydrolysis method gave satisfactory results without the need for separate analyses of tryptophan and cysteine, provided proteins were reduced and carboxymethylated prior to hydrolysis.     

Recovery of tryptophan in peptides and proteins by high-temperature and short-term acid hydrolysis in the presence of phenol

The addition of 3% (w/v) phenol to 6 M HCl largely prevented the destruction of tryptophan during rapid hydrolysis of peptides and proteins at 166 degrees C for 25 min or at 145 degrees C for 4 h. This hydrolysis procedure was advantageous for amino acid microanalysis using conventional high-performance liquid chromatography with a precolumn derivatization technique. The recovery of tryptophan from proteins was at least 80%. The addition of phenol also improved the recovery of methionine and carboxymethylcysteine. The amount of tryptophan in proteins electroblotted onto a polyvinylidene difluoride membrane was determined by this method.     

Fluorometric assay for intestinal peptidases

A fluorometric assay for intestinal peptidases has been developed. Amino acids liberated by hydrolysis are estimated by use of l-amino-acid oxidase, peroxidase, and the fluorogenic reagent p-hydroxyphenylacetic acid, which yields a highly fluorescent compound on oxidation. During development of fluorescence, continued hydrolysis of peptides by peptidases which contaminate available preparations of l-amino-acid oxidase is prevented by the use of two inhibitors, 1,10-phenanthroline and p-hydroxymercuribenzoate. The assay is at least 10 times more sensitive than comparable spectrophotometric methods which employ the potentially carcinogenic chromogen o-dianisidine for detection of amino acids.     

Small peptides,a life-long store of amino acid in adult Drosophila and Calliphora

A sizable component of small peptide is probably an important store of amino acid in Calliphora and Drosophila. Quantitative amino acid analysis of the peptides in adult male Calliphora erythrocephala and Drosophila subobscura show that their composition is very similar. It is shown in Calliphora that the amount of peptide present in adults varies with age and feeding regime. Further, the peptide composition of the haemolymph indicates that the peptides are stored intracellularly. However, there is evidence which suggests that their hydrolysis takes place in the haemolymph, thus providing the whole fly with free amino acid and the haemolymph with osmotic stability.     

A manual subtractive end-group determination of oligopeptides using fluorescamine or o-phthalaldehyde

A new method for the end-group determination of peptides using the fluorogenic reagents fluorescamine or o-phthalaldehyde is described. The method is based on the property that the derivatives of the N-terminal amino group of peptides formed in solution after reaction with either reagent are resistant to acid hydrolysis. The N-terminal amino acid can be determined by simply comparing the amino acid analysis of the original peptide with the fluorescent derivative of the peptide. In general, the decrease of the N-terminal residue in the reacted peptides in 80–90% with fluorescamine and more than 90% with o-phthalaldehyde. Any N-terminal amino acid, with the exception of proline, can thus be determined.     

Development of an acid hydrolysis method with high recoveries of tryptophan and cysteine for microquantities of protein.

High recoveries of tryptophan and cysteine were achieved by 12.5 min of hydrolysis with mercaptoethanesulfonic acid vapor. Proteins (1-100 micrograms) were modified by vapor-phase S-pyridylethylation before hydrolysis. The modified proteins were hydrolyzed with the vapor of 2.5 M mercaptoethanesulfonic acid at 176 degrees C. This method promoted efficient hydrolysis of the peptide bonds in proteins and resulted in high recoveries of both tryptophan and cysteine, of 90% or greater, in addition to the other amino acids.     

Interference of ethanol in the determination of tryptophan by the method of spies and chambers.

Ethanol interference with the Spies and Chambers method (1–3) for the determination of tryptophan is shown in the results with cereal proteins. This negative interference of ethanol was confirmed by use of free tryptophan under the same conditions. The extent of ethanol interference varies with the sample. It is postulated that ethanol inhibits the tryptophan-p-dimethylaminobenzaldehyde (DAB) reaction by alkylation of the amino acid and possibly to a lesser extent, by acetal formation with DAB. A method for the removal of ethanol from samples before tryptophan determination is suggested.     

毛细管水解及反相高效液相色谱分析蛋白质的氨基酸组成

 毛细管水解及反相高效液相色谱分析蛋白质的氨基酸组成陈平,梁宋平（湖南师范大学生物研究所,长沙４１０００６）氨基酸组成的分析是阐明蛋白质和多肽化学特性的基础,在蛋白质与多肽的氨基酸组成分析中,常采用对水解管反复充氮并抽真空的方法使蛋白质和多肽在隔绝氧气...     

Direct amino acid analysis of proteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel

The band of appropriate proteins (basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, interleukin 2, and human leukocyte interferon alpha A) on a polyvinylidene difluoride (PVDF) membrane, which was electroblotted from sodium dodecyl sulfate (SDS)-polyacrylamide gel and then stained with Coomassie blue R-250, was cut out and directly hydrolyzed in HCl in the presence of thioglycolic acid for amino acid analysis. The analytical values agreed with those expected with recoveries of 29-47%, except that the value for tryptophan was very low or scarcely detected. This method was applied to the identification of human growth hormone (hGH) in a partially purified preparation. The amino acid composition of the band corresponding to about 2 micrograms of hGH agreed with the theoretical values. These results indicate that the band on the PVDF membrane can be directly hydrolyzed for amino acid analysis and that the method can be used for partially purified proteins separated using SDS-polyacrylamide gel electrophoresis.     

Comparative chemical studies of the polyhedral proteins of the nuclear polyhedrosis viruses of Bombyx mori and Galleria mellonella

Amino and carboxyl terminal groups, amino acid composition, and peptide maps of polyhedral proteins of the nuclear polyhedrosis viruses (NPV) of Bombyx mori and Galleria mellonella were investigated. It is shown that both the proteins have a tyrosine residue as their carboxyl terminal group and no amino terminal group. Amino acid compositions of the proteins are similar. The proteins are found to have 242 residues. From the amino acid composition, a molecular weight of 28,000 was calculated. The tryptic peptide maps of both the proteins differed only in a few peptides.It is inferred that the polyhedral proteins of B. mori and G. mellonella NPV have a closely similar primary structure.     

The primary structure of the human pancreatic secretory trypsin inhibitor: Amino acid sequence of the reduced S-aminoethylated protein

The amino acid sequence of human pancreatic secretory trypsin inhibitor [Pubols, M. H., Bartelt, D. C., and Greene, L. J. (1974) J. Biol. Chem., 249, 2235–2242] was determined by a combination of selective trypsin and chymotrypsin hydrolysis reactions on the S-2-aminoethylcysteinyl inhibitor and conventional methods (subtractive Edman degradation and exopeptidase hydrolysis) for sequence determination of small peptides. The peptides were ordered on the basis of the identification of the amino- and carboxyterminal residues of the products at each stage of the degradation procedure. The sequence determination was carried out on a mixture of chromatographic forms present in both tissue and pancreatic juice which are identical in amino acid composition, aminoterminal residues, molecular weight, and specific activity, but differ only in asparagine content and susceptibility to enzymatic hydrolysis. The amino acid sequence of the human inhibitor corresponding to chromatographic form A₃ has been shown to be NH₂

.     

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.