Rapid removal of acetimidoyl groups from proteins and peptides. Applications to primary structure determination.

Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies.     

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.     

Heat capacities of solid poly(amino acid)s. II. The remaining polymers

In the first paper heat capacities C_p, of polyglycine, poly(L -alanine), and poly (L -valine) were analyzed using approximate group vibrations and fitting the C_p contributions of the skeletal vibrations to a two-parameter Tarasov function. In this second paper all other poly (amino acid) s are similarly analyzed. Heat capacities were measured by differential scanning calorimetry in the temperature range of 230–390 K for poly(L -leucine), poly(L -serine), poly (sodium-L -aspartate), poly(sodium-L -glutamate), poly(L -asparagine), poly(L -phenylalanine), poly(L -tyrosine), poly(L -methionine), poly (L -tryptophane), poly(L -proline), poly(L -lysine · HBr), poly(L -histidine), poly(L -histidine- HCl), and poly(L -arginine · HCl). Good agreement exists between experiment and calculation. Predictions of heat capacities were made for all not-measured poly (amino acid) s. Enthalpies, entropies, and Gibbs functions for the solid state have been derived. © 1993 John Wiley & Sons, Inc.     

Reactions of hydroxyamino acids during hydrochloric acid hydrolysis

Chlorosubstitution reactions occur readily during HCl hydrolysis of delta- and epsilon-hydroxynorleucines (Hnle), the products of deamination of poly-L-lysine by nitrite at low pH. During amino acid analysis, chloronorleucines elute as new peaks after delta- and epsilon-Hnle. To determine if other hydroxyamino acids undergo similar changes during hydrolysis, they were subjected individually to HCl hydrolysis conditions with and without added phenol. Amino acid analyses indicated that terminal hydroxy groups on linear side chains undergo reactions during HCl hydrolysis; the products appear as new peaks which may be chloroderivatives. In contrast, no new peaks are observed in HCl hydrolysates of delta-hydroxylysine or amino acids with beta-hydroxy groups (beta-hydroxynorvaline, serine, and threonine). Phenol did not protect linear amino acids from reactions during HCl hydrolysis but did prevent loss of the cyclic amino acids tyrosine, hydroxyproline, and 3,4-dihydroxyphenylalanine. Although the gamma-hydroxy group of homoserine would be expected to undergo reaction, HCl catalyzes its cyclization to form homoserine lactone instead.     

Micro-determination of amino acid composition of proteins electroblotted onto polyvinylidene difluoride membranes

A method for determination of amino acid composition of proteins separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes is described. A single blotted band containing 50 to 200 pmoles of protein was cut out and submitted to acid hydrolysis with HCl followed by derivatization with phenylisothiocyanate. The amino acid derivatives were separated by reverse phase high-performance liquid chromatography. Bovine serum albumin, lysozyme, myoglobin, ovalbumin, soybean trypsin inhibitor and carbonic anhydrase were analyzed; the results revealed a good correspondence with reported values. This can be considered an analytical method to determine the amino acid composition of samples from microquantities of protein mixtures, particularly in those cases in which SDS-polyacrylamide gel electrophoresis is the most suitable separation system.     

Determination of methionine sulfoxide in protein and food by hydrolysis with p-toluenesulfonic acid

Methionine sulfoxide in peptides and proteins was determined by use of 3 N p-toluenesulfonic acid as a hydrolyzing agent. Samples were hydrolyzed at 110 degrees C for 22 h in an evacuated sealed tube and analyzed for amino acid content. Amino acid analysis showed that the recovery of methionine sulfoxide from a synthetic peptide and its mixture with proteins was consistently better than 90%. The recovery of all other amino acids except tryptophan was complete, and was similar to that observed after hydrolysis with 6 N HCl. The presence of carbohydrates had no effect on the yield. Thus, the present procedure can be used for general and simultaneous determination of methionine sulfoxide as well as other amino acids in proteins.     

Convenient synthesis of bifunctional tetraaza macrocycles.

A convenient synthesis of 4-nitrobenzyl-substituted macrocyclic tetraamines and their conversion to bifunctional poly(amino carboxylate) chelating agents is described. Cyclization of (4-nitrobenzyl)-ethylenediamine with appropriate BOC-protected amino disuccinimido esters in dioxane at 90 degrees C resulted in the formation of 12- and 14-membered ring diamides in 40% and 44% yield, respectively. A 12-membered macrocyclic triamide was also prepared in 44% yield by cyclization of N-(2-aminoethyl)-4-nitrophenylalaninamide with disuccinimidyl N-(tert-butoxycarbonyl)iminodiacetate. Deprotection (HCl/dioxane) and reduction with borane gave the substituted macrocyclic amines which were then alkylated with either bromoacetic acid or tert-butyl bromoacetate. Preparation of the isothiocyanate derivatives and 14C labeled chelating agents are described. Attempts to prepare a 9-membered macrocyclic diamide using this cyclization technique resulted instead in a 20% yield of a 10:1 mixture of isomeric fused 5,6 ring acylamidines. Deprotection (HCl/dioxane) and reduction with borane gave a substituted piperazine derivative in 55% yield.     

Amino acid analysis on polyvinylidene difluoride membranes

A procedure for the amino acid analysis of proteins electrotransferred to polyvinylidene difluoride (PVDF) membranes is described. The proteins are first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a PVDF membrane. After staining with Coomassie brilliant blue, the visualized protein bands are excised from the membrane. Each band is placed in a vial and subjected to gas-phase hydrolysis in 6 N HCl in a vacuum desiccator at 110 degrees C. The amino acids are extracted from the membrane into 0.1 N HCl/30% CH3OH and analyzed by reverse-phase HPLC using postcolumn o-phthalaldehyde-derivatizing reagent. The method was shown to give reproducible and reasonably accurate compositions for several proteins, as well as to provide an estimate of protein content. As little as 10 pmol of a 67-kDa protein can be determined.     

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.     

圈养大熊猫乳中氨基酸组成的研究

为了认识大熊猫乳中氨基酸的组成特点,将大熊猫乳经6 mol/L盐酸水解后,用高效液相色谱测定了不同泌乳阶段的20个乳样的氨基酸组成.结果显示,大熊猫初乳中含量最高的氨基酸是谷氨酸,约为5.2 g/L;常乳和干乳期乳腺分泌物中含量最高的是脯氨酸;总氨基酸含量在泌乳2～10 d内变化不明显,平均值约为34 g/L;干乳期乳中氨基酸含量变化显著.     

Synthesis and antitumor activity of arginine–glucosamine conjugate

Using d-glucosamine hydrochloride (GlcNH₂·HCl) as starting material, a new amino acid sugar conjugate, arginine–glucosamine (Arg–GlcNH₂), was synthesized and characterized by infrared spectroscopy, ¹³C NMR and element analysis. Its cytotoxicity in vitro was evaluated by MTT assay. The inhibition ratio against human hepatoma cell SMMC-7721 was higher than that of GlcNH₂·HCl. This effect was accompanied by a marked increase in the proportion of S cells as analyzed by flow cytometry. In addition, SMMC-7721 cells treated with Arg–GlcNH₂ resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis. The manner of Arg–GlcNH₂ cytocidal activity was concluded to be apoptosis.     

Gustatory responses to amino acids in the chorda tympani nerve of C3H mice

Integrated neural responses to various amino acids were recorded from the chorda tympani (facial) nerve in C3H mice. The basic amino acids hydrochlorides L-Arg-HCl and L-Lys-HCl evoked large magnitude integrated taste responses, similar to that for NaCl, and had estimated electrophysiological thresholds of 0.0001 M. No significant difference was indicated between the response magnitudes for the L- and D-forms of the basic amino acid hydrochlorides; however, responses to the basic amino acid hydrochlorides cross-adapted with NaCl. Responses to neutral L-amino acids (Ser, Ala, Gly), which taste sweet to humans, showed higher thresholds (>0.0003 M), similar to that for sucrose, and did not cross-adapt with basic amino acid hydrochlorides or with NaCl. Responses to the neutral amino acids L-Ser and L-Ala were larger than those to their D-amino acid enantiomers. The acidic amino acids L-Asp and L-Glu showed concentration-response functions different from that for HCl. Both acidic amino acids were more stimulatory than HCl at the same pH, although the responses to them were cross-adapted by HCl, indicating a pH effect. A comparison of the stimulatory effectiveness among amino acid derivatives and analogues suggested that the alpha- amino group is essential for the stimulatory effectiveness of neutral amino acids.      

Regulation of Amino Acid Intake in the Rat: Lysine Preference with Gluten Diets of Varying Lysine Content

Weanling rats were offered a choice of two diets, varying only in lysine content. The preference test was performed between a 20% gluten diet supplemented with 0.5% lysine HCl and a lysine-deficient amino acid mixture, 20% gluten or 20% gluten plus 0.2% lysine HCl diet, and was done between a 20% gluten diet and a 20% gluten diet containing 3, 5 or 7% lysine · HCl. Weight gain and food intake were not significantly different among all the self-selecting groups, and these values were almost the same as those in rats fed only the 20% gluten supplemented 0.5% lysine · HCl diet which obtained maximal growth. The lysine intake of the combination groups ranged from 67 to 240 mg per day (0.62-2.35% of consumed food). It was demonstrated that rats exhibit a definite ability to regulate lysine intake, and they select sufficient lysine to meet their requirements for this amino acid. The self-selection technique may be useful as a method to determine the requirements for amino acids.     

The direct hydrolysis of proteins containing tryptophan on polyvinylidene difluoride membranes by mercaptoethanesulfonic acid in the vapor phase.

A procedure for the amino acid analysis of polypeptides that contain tryptophan on polyvinylidene difluoride membranes is described. Lysozyme, carbonic anhydrase, phytochrome, and ovalbumin were tested. The protein, which was separated from others by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was blotted from the gel onto a polyvinylidene difluoride membrane and directly hydrolyzed by 3 N mercaptoethanesulfonic acid vapor in a vacuum at 176 degrees C for 25 min. The hydrolysate was extracted with 0.1 N HCl and 30% methanol and used for amino acid analysis. The tested proteins were adequately hydrolyzed, and the recovery of tryptophan was very efficient.     

吊瓜籽中氨基酸质量分数的测定

样品吊瓜籽经酸水解处理,应用氨基酸分析仪进行分析,结果表明,吊瓜籽含所有常规17种氨基酸,质量分数高达23.28%,其中谷氨酸(G lu)和精氨酸(Agr)质量分数达4.39%和3.88%。     

麦苗中氨基酸测定与评价

用日立L-8800型氨基酸自动分析仪测定了麦苗中的氨基酸含量.结果表明麦苗中18种氨基酸总量、必需氨基酸及药效氨基酸含量较高,提示麦苗在营养学和医学上都有很高的研究价值.     

Ionization of adenine derivatives: EPR and ENDOR studies of X-irradiated adenine.HCl.1/2H2O and adenosine.HCl.

Following X irradiation of adenine.HCl.H2O at 10 K, evidence for five distinct radical products was present in the EPR/ENDOR. (In both adenine.HCl.1/2H2O and adenosine.HCl, the adenine base is present in a cationic form as it is protonated at N1). From ENDOR data, radical R1, stable at temperatures up to 250 K, was identified as the product of net hydrogen loss from N1. This product, evidently formed by electron loss followed by proton loss, is equivalent to the radical cation of the neutral adenine base. Radical R2, unstable at temperatures above 60 K, was identified as the product of net hydrogen addition to N3, and evidently formed by electron addition followed by proton addition. Radicals R3-R5 could not be identified with certainty. Similar treatment of adenosine.HCl provided evidence for six identifiable radical products. Radical R6, stable to ca. 150 K, was identified as the result of net hydrogen loss from the amino group, and evidently was the product of electron loss followed by proton loss. Radical R7 was tentatively identified as the product of net hydrogen addition to C4 of the adenine base. Radical R8 was found to be the product of net hydrogen addition to C2 of the adenine base, and R9 was the product of net hydrogen addition to C8. Radical R10 was identified as the product of net hydrogen abstraction from C1' of the ribose, and R11 was an alkoxy radical formed from the ribose. With the exception of R11, all products were also found following irradiation at 65 K. Only radical R8 and R9 were stable at room temperature. Most notable is the different deprotonation behavior of the primary electron-loss products (radical R1 vs. R6) and the different protonation behavior of the primary electron-gain products (radical R2 vs. no similar product in adenosine.HCl). The major structural difference in the two crystals is the electrostatic environment of the adenine base. Therefore, this study provides further evidence that environmental influences are important in determining proton transfer processes.     

Occurrence of ditaurobilirubin, bilirubin conjugated with two moles of taurine, in the gallbladder bile of yellowtail, Seriola quinqueradiata

An unknown bilirubin conjugate was detected by HPLC analysis in the bile of yellowtail, Seriola quinqueradiata. The retention time of this unknown conjugate was identical with that of authentic ditaurobilirubin, a bilirubin conjugated with 2 mol of taurine. The retention times of the ethyl anthranilate diazo derivatives of both bilirubin conjugates were the same. The azo derivatives of this unknown conjugate were purified by Amberlite XAD-2 treatment and preparative TLC and hydrolyzed with 6 N HCl at 105 degrees C for 12 h. The hydrolyzates obtained were analyzed with an amino acid analyzer. The ninhydrin-positive substance in the hydrolyzates was purified by column chromatography and analyzed by TLC and mass spectrometry. The results of these analyses indicated that this ninhydrin-positive substance was taurine. Therefore, this unknown bilirubin conjugate was identified as ditaurobilirubin.     

羊胎素中氨基酸测定与评价

用日立L-8800型氨基酸自动分析仪测定了羊胎素中的氨基酸含量。结果表明羊胎素中18种氨基酸总量、必需氨基酸及药效氨基酸含量较高,提示羊胎素在营养学和医学上都有很高的研究价值。     

Oxidation of tryptophan to oxindolylalanine by dimethyl sulfoxide-hydrochloric acid. Selective modification of tryptophan containing peptides

Tryptophan is readily oxidized to oxindolylalanine (2-hydroxytryptophan) in good yield on treatment in acetic acid solution with a mixture of dimethyl sulfoxide (DMSO) and concentrated aqueous HCl at room temperature. Other sulfoxides can be used in combination with HCl; for example, methionine sulfoxide reacts with an equimolar amount of tryptophan to give high yields of methionine and oxindolylalanine. Methionine and cysteine are quantitatively oxidized by DMSO/HCl to methionine sulfoxide and cystine, respectively. The tryptophan containing peptides LRF (luteinizing hormone-releasing factor), somatostatin, valine-gramicidin A and ACTH 1-24 were each treated with the DMSO/HCl reagent in acetic acid solution and the corresponding oxindolylalanine-derivatives isolated in over 90% yield after chromatography. The identity and purity of the derivatives were established on the basis of ultraviolet spectral characteristics and quantitative amino acid analysis of the oxindolylalanine content of acid hydrolyzates of the oxidized peptides with 3N-p-toluenesulfonic acid at 110 degrees for 24 h. The results indicate that modification of tryptophan peptides with DMSO/HCl provides a useful procedure, which seems superior to previously used reagents. In addition, the method could be well applied to other indoles of biological and pharmacological interest.