Variations in aspartic acid racemization in uniformly preserved plants about 11000 years old

Amino acid racemization forms a basis for determining the chronology and paleotemperature of old plant constituents. Disparity in the extent of aspartic acid racemization was found in different taxa of plants subjected to the same environmental history and found in close proximity within an ancient packrat midden. One taxon showed different rates of aspartic acid racemization in two different anatomical sites. Temperature, pH and time being virtually identical in this one micro-environment within the midden, the differences in racemization rates may have been ultimately derived from physiological variants among the plants. Thus, at least, aspartic acid racemization data should be used selectively.     

Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro‐organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 °C and 1–2 years for 3 km depth and 54 °C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 °C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro‐organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.     

Investigations of in vivo methionine racemization in mammalian tissues

The racemization rate of methionine at pH 7.4 and 100 degrees C was determined to be similar to that of aspartic acid. However, analyses of dentin and ocular lens nucleus samples from known age Rhesus monkeys showed that unlike aspartic acid, methionine does not undergo in vivo racemization in mammalian proteins.     

Estimating narwhal (Monodon monoceros) age using tooth layers and aspartic acid racemization of eye lens nuclei

Counting growth-layer groups (GLGs) in teeth is one of the most precise and widely accepted methods for aging marine mammals. Male narwhals have a large erupted tusk that can be used for aging, but this tusk is often difficult or expensive to obtain from hunters and most females do not display the tusk; thus, alternative methods for narwhal aging are needed. In this study, we aged narwhals by counting annual GLGs in embedded tusks and by measuring the change in the ratio of D- and L-enantiomers of aspartic acid in the eye lens nucleus that occurs as the animal ages (the aspartic acid racemization [AAR] technique). Absolute age estimates were estimated for seven tusks aged ≤15 yr. Estimated age was a significant predictor of aspartic acid D/L ratios with a racemization rate (K_asp) of 9.72 × 10⁻⁴/year ± 2.28 × 10⁻⁴ and a (D/L)₀ of 3.46 × 10⁻² ± 1.78 × 10⁻³ (r² = 0.74). Results from our study, which included more younger GLG-aged animals than previously evaluated, confirms AAR can be used to generate age estimates for narwhals.     

Collagen turnover in normal and degenerate human intervertebral discs as determined by the racemization of aspartic acid

Knowledge of rates of protein turnover is important for a quantitative understanding of tissue synthesis and catabolism. In this work, we have used the racemization of aspartic acid as a marker for the turnover of collagen obtained from healthy and pathological human intervertebral disc matrices. We measured the ratio of the d- and l-isomers in collagen extracted from these tissues as a function of age between 16 and 77 years. For collagen taken from healthy discs, the fractional increase of d-Asp was found to be 6.74 x 10(-4)/year; for degenerate discs, the corresponding rate was 5.18 x 10(-4)/year. Using the racemization rate found previously for the stable population of collagen molecules in dentin, we found that the rate of collagen turnover (k(T)) in discs is not constant but rather a decreasing function of age. The average turnover rate in normal disc between the ages of 20 and 40 is 0.00728 +/- 0.00275/year, and that between the ages of 50 and 80 is 0.00323 +/- 0.000947/year, which correspond to average half-lives of 95 and 215 years, respectively. Turnover of collagen from degenerate discs may be more rapid than that found for normal discs; however, statistical analysis leaves this point uncertain. The finding of a similar correlation between the accumulation of d-Asp and that of pentosidine for three normal collagenous tissues further supports the idea that the accumulation of pentosidine in a particular tissue can, along with the racemization of aspartic acid, be used as a reliable measure of protein turnover.     

Predicting protein decomposition: the case of aspartic-acid racemization kinetics

The increase in proportion of the non-biological (D-) isomer of aspartic acid (Asp) relative to the L-isomer has been widely used in archaeology and geochemistry as a tool for dating. the method has proved controversial, particularly when used for bones. The non-linear kinetics of Asp racemization have prompted a number of suggestions as to the underlying mechanism(s) and have led to the use of mathematical transformations which linearize the increase in D-Asp with respect to time. Using one example, a suggestion that the initial rapid phase of Asp racemization is due to a contribution from asparagine (Asn), we demonstrate how a simple model of the degradation and racemization of Asn can be used to predict the observed kinetics. A more complex model of peptide bound Asx (Asn + Asp) racemization, which occurs via the formation of a cyclic succinimide (Asu), can be used to correctly predict Asx racemization kinetics in proteins at high temperatures (95-140 degrees C). The model fails to predict racemization kinetics in dentine collagen at 37 degrees C. The reason for this is that Asu formation is highly conformation dependent and is predicted to occur extremely slowly in triple helical collagen. As conformation strongly influences the rate of Asu formation and hence Asx racemization, the use of extrapolation from high temperatures to estimate racemization kinetics of Asx in proteins below their denaturation temperature is called into question. In the case of archaeological bone, we argue that the D:L ratio of Asx reflects the proportion of non-helical to helical collagen, overlain by the effects of leaching of more soluble (and conformationally unconstrained) peptides. Thus, racemization kinetics in bone are potentially unpredictable, and the proposed use of Asx racemization to estimate the extent of DNA depurination in archaeological bones is challenged.     

Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.

Microwave energy represents an efficient manner to accelerate both the deprotection and coupling reactions in 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). Typical SPPS side reactions including racemization and aspartimide formation can occur with microwave energy but can easily be controlled by routine use of optimized methods. Cysteine, histidine, and aspartic acid were susceptible to racemization during microwave SPPS of a model 20mer peptide containing all 20 natural amino acids. Lowering the microwave coupling temperature from 80 degrees C to 50 degrees C limited racemization of histidine and cysteine. Additionally, coupling of both histidine and cysteine can be performed conventionally while the rest of the peptide is synthesized using microwave without any deleterious effect, as racemization during the coupling reaction was limited to the activated ester state of the amino acids up to 80 degrees C. Use of the hindered amine, collidine, in the coupling reaction also minimized formation of D-cysteine. Aspartimide formation and subsequent racemization of aspartic acid was reduced by the addition of HOBt to the deprotection solution and/or use of piperazine in place of piperidine.     

The influence of extrusion on loss and racemization of amino acids

Summary. The influence of the operation conditions (temperature and residence time) of a thermic treatment on the total amount (free and protein-bound) of amino acid enantiomers of dry fullfat soya was investigated. Total amino acid content was determined using conventional ion-exchange amino acid analysis of total hydrolysates and chiral amino acid analysis was performed by HPLC after precolumn derivatization with o-phthaldialdehyde and 1-thio-β-D-glucose tetraacetate. Contrary to corn that was investigated previously, notable racemization was detected even at lower temperatures. At 140 °C the ratio of the D-enantiomer was 0.87% for glutamic acid, 2.81% for serine, and 1.92% for phenylalanine; at 220 °C the ratios of the D-enantiomer of the above amino acids were 1.43, 4.61, and 4.68%, respectively. The concentration of several L-amino acids decreased. At 220 °C there was 10% less L-glutamic acid, 17% less L-serine, 5% less L-phenylalanine, 6.6% less L-aspartic, acid and 21% less L-lysine than in the control; their loss can be assigned to different degrees of L – D conversion. While nearly complete transformation of L-phenylalanine can be attributed to racemization, the main cause of the loss of L-lysine is not racemization. The treatments in the same order of magnitude resulted in the formation of more D-amino acids and greater extent of racemization of amino acids in fullfat soya than that of maize. Authors’ address: J. Csapó, Faculty of Animal Science, Institute of Chemistry, University of Kaposvár, Guba S. u. 40., H-7400 Kaposvár, Hungary     

Total hydrolysis of proteins with strongly reduced racemization of amino acids

A new method has been devised for the complete hydrolysis of proteins with an extremely low level of racemization of amino acids. Proteins are incubated in 10 M HCl at a low temperature to obtain partial hydrolysis. They are then incubated with pronase and finally with leucine aminopeptidase and peptidyl-D-amino-acid hydrolase from Loligo vulgaris. The proposed method ensures the total hydrolysis of either purified proteins or proteins contained in a crude homogenate of animal or vegetable tissue. In both cases, the racemization of amino acids (expressed as rate of D form/D + L form X 100) was lower than 0.015% for aspartic acid and lower than 0.01% for other amino acids. D-Amino acids released from peptides or proteins were estimated with enzymatic methods based on the use of octopus D-aspartate oxidase or hog kidney D-amino acid oxidase; with these enzymes, 0.05 nmol of a D-amino acid was determined in the presence of up to 20 mumols of a mixture of L-amino acids (ratio %D/D + L = 0.00025). The method allows the determination of D-amino acids either in tissues in which they are present in high concentrations (as human cataract lenses, tooth enamel, etc.) or in those with low enantiomer content (as brain, erythrocytes, etc.). Using the method described, we hydrolyzed several synthetic peptides consisting of D- and L-amino acids and determined the amount of D-amino acids. In addition, we totally hydrolyzed all the nuclear proteins of human cataractous lenses. The amount of D-aspartic acid was 0.026 mumols/mg in lenses of women aged between 71 and 76 years and 0.0256 mumols/mg in lenses of men aged between 55 and 72 years. The D-aspartic acid measured corresponds to about 12% with respect to total aspartic acid.     

Base-catalyzed racemization of 3-O-acyloxazepam

Enantiomers of 3-O-acyloxazepam (oxazepam 3-acetate; OXA) underwent base-catalyzed hydrolysis and racemization. Kinetics of reaction products formed from an OXA enantiomer in buffered and unbuffered alkaline solutions were analyzed by chiral stationary phase high-performance liquid chromatography. Racemization occurred with varying rates in aqueous solutions with pH ranging from 7.5 to 14. Racemization mechanism was studied by the dependence of rates of hydrolysis and racemization on temperature and pH. Mass spectral analysis of racemization products derived from an OXA enantiomer in a deuterated solvent indicated that racemization was accompanied by a proton exchange with the solvent. The results indicated that a base-catalyzed keto-enol tautomerism between the C2-carbonyl group and the C3 carbon was responsible for the observed racemization. © 1994 Wiley-Liss, Inc.

1 This article is a US Goverment work and, as such, is in the public domain in the United States of America.

     

Aggrecan turnover in human intervertebral disc as determined by the racemization of aspartic acid

We have used the racemization of aspartic acid as a marker for the "molecular age" of aggrecan components of the human intervertebral disc matrix (aggregating and non-aggregating proteoglycans as well as the different buoyant density fractions of aggrecan). By measuring the D/L(Asp) ratio of the various aggrecan species as a function of age and using the values of the racemization constant, k(i), found earlier for aggrecan in articular cartilage, we were able to establish directly the relative residence time of these molecules in human intervertebral disc matrix. For A1 preparations taken from normal tissue, turnover rates of 0.059 +/- 0.01 and 0.063 +/- 0.01/year correspond to half-life values of 12 +/- 2.0 and 11.23 +/- 1.9 years for nucleus pulposus and annulus fibrosus, respectively; the turnover rates of 0.084 +/- 0.022 and 0.092 +/- 0.034/year for degenerate tissue correspond to half-life values of 8.77 +/- 2.2 and 8.41 +/- 2.8 years, suggesting increased rate of removal of small aggrecan fragments. For the large monomer, fraction A1D1, turnover is 0.13 +/- 0.04/year, corresponding to a half-life of 5.56 +/- 1.58 years, similar to 3.4 years in human articular cartilage. For the binding region (A1D6), turnover is 0.033 +/- 0.0012/year, corresponding to a half-life of 21.53 +/- 0.6 years, similar to 23.5 years in articular cartilage. A1 preparations from nucleus pulposus contain a lower proportion of aggregating proteoglycans as compared with annulus fibrosus, suggesting increased proteolytic modification in the nucleus pulposus. D/L(Asp) values in aggregating and non-aggregating proteoglycans of a 24-year-old individual show similar results, suggesting that the non-aggregating molecules are synthesized initially as aggregating proteoglycans, which thereafter undergo cleavage and detachment from hyaluronan.     

Radiolysis,racemization, and the origin of optical activity

An investigation has been undertaken to determine whether ionizing radiation might engender racemization (radioracemization) of optically active amino acids, along with their well-known radiolysis. We have exposed a number of solid and dissolved optically active amino acids to the ionizing radiation from a 3000-Ci ⁶⁰Co γ-ray source for periods of time which would engender substantial, but not total radiolysis. γ-Ray doses which caused 55–68% radiolysis of solid amino acids typically engendered 2–5% racemization. Aqueous solutions of the sodium salts of amino acids which underwent 53–66% radiolysis typically showed 5–11% racemization. The corresponding hydrochloride salts in aqueous solution, however, underwent little or no racemization. In aqueous solution both percentage degradation and percentage racemization were approximately proportional to γ-ray dosage within the range employed (1–36 × 10⁶ rads). Mechanisms for the radioracemization of amino acids in the solid state and as dissolved sodium salts are proposed, and the absence of racemization for dissolved hydrochloride salts is rationalized. Implications of these observations with regard to the origin of optical activity by the Vester-Ulbricht β-decay mechanism are discussed, as are their implications regarding the use of diagenetic racemization rates of ancient amino acid samples as criteria for geochronological and geothermometric calculations.     

Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation

Aspartyl and asparaginyl deamidation, isomerization, and racemization reactions have been studied in synthetic peptides to model these spontaneous processes that alter protein structure and function. We show here that the peptide L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala undergoes a rapid deamidation reaction with a half-life of only 1.4 days at 37 degrees C, pH 7.4, to give an aspartyl succinimide product. Under these conditions, the succinimide product can further react by hydrolysis (half-time, 2.3h) and by racemization (half-time, 19.5 h). The net product of the deamidation reaction is a mixture of L- and D-normal aspartyl and beta-transpeptidation (isoaspartyl) hexapeptides. Replacement of the asparagine residue by an aspartic acid residue results in a 34-fold decrease in the rate of succinimide formation. Significant racemization was found to accompany the deamidation and isomerization reactions, and most of this could be accounted for by the rapid racemization of the succinimide intermediate. Replacement of the glycyl residue in the asparagine-containing peptide with a bulky leucyl or prolyl residue results in a 33-50-fold decrease in the rate of degradation. Peptide cleavage products are observed when these Asn-Leu and Asn-Pro-containing peptides are incubated. Our studies indicate that both aspartic acid and asparagine residues may be hot spots for the nonenzymatic degradation of proteins, especially in cells such as erythrocytes and eye lens, where these macromolecules must function for periods of about 120 days and 80 years, respectively.     

Role of racemization in optically active drugs development

U.S. Food and Drug Administration issues certain guidelines for marketing of optically active drugs as some enantiomers racemize into human body, leading to the generation of other antipodes, which may be toxic or ballast to the human beings. Moreover, racemization reduces the administrated dosage concentration as optically active enantiomer converted into its inactive counter part. Therefore, the study of racemization of such type of drugs is an important and urgent need of today. This article describes in vitro and in vivo racemization of optically active drugs. The racemization process of various optically active drugs has been discussed considering the effect of different variables i.e. pH, temperature, concentration of the drug, ionic concentration, etc. Attempts have also been made to discuss the mechanisms of racemization. Besides, efforts have been made to suggest the safe dosages of such type of drugs too.     

Substitution and racemization of 3-hydroxy- and 3-alkoxy-1,4-benzodiazepines in acidic aqueous solutions

Oxazepam (OX), 3-O-methyloxazepam, 3-O-ethyloxazepam, temazepam (TMZ), 3-O-methyltemazepam, and 3-O-ethyltemazepam underwent acid-catalyzed nucleophilic substitution reaction (hydrolysis) in an acetonitrile–oxygen-18 water mixture to form either OX or TMZ in which the 3-hydroxyl group was either partially or fully labeled with an oxygen-18 atom. The dependence of the hydrolysis rates on solvent composition, temperature, ionic strength, and in deuterated solvent was studied by reversed-phase high-performance liquid chromatography (HPLC). The rates of racemization of enantiomeric compounds in acidic aqueous solutions were studied by both spectropolarimetry and chiral stationary phase HPLC. In acetonitrile: 2.5 M H₂SO₄ (4:1, v/v) at 50°C, enantiomers of OX and TMZ underwent racemization at rates ≥40-fold faster than the rates of hydrolysis. Enantiomeric 3-O-alkyl derivatives of OX and TMZ in acidic aqueous solutions did not themselves undergo racemization and it was their hydrolysis products (either OX or TMZ) that underwent racemization. © 1995 Wiley-Liss, Inc.     

Serine hydroxymethyltransferase: mechanism of the racemization and transamination of D- and L-alanine

The reaction specificity and stereochemical control of Escherichia coli serine hydroxymethyltransferase were investigated with D- and L-alanine as substrates. An active-site H228N mutant enzyme binds both D- and L-alanine with Kd values of 5 mM as compared to 30 and 10 mM, respectively, for the wild-type enzyme. Both wild-type and H228N enzymes form quinonoid complexes absorbing at 505 nm by catalyzing the loss of the alpha-proton from both D- and L-alanine. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The relative rates of these reactions are quinonoid formation greater than alpha-proton solvent exchange greater than racemization greater than transamination. The observation that the rate of quinonoid formation with either alanine isomer is an order of magnitude faster than solvent exchange suggests that the alpha-protons from both D- and L-alanine are transferred to base(s) on the enzyme. The rate of racemization is 2 orders of magnitude slower than the formation of the quinonoid complexes. This latter difference in rate suggests that the quinonoid complexes formed from D- and L-alanine are not identical. The difference in structure of the two quinonoid complexes is proposed to be the active-site location of the alpha-protons lost from the two alanine isomers, rather than two orientations of the pyridoxal phosphate ring. The results are consistent with a two-base mechanism for racemization.     

Purification and characterization of a protein containing D-aspartic acid in bovine lens

A protein containing biologically uncommon D-aspartic acid (DAsp) was extracted with 60% EtOH from the water-insoluble fraction of bovine lens. The protein was purified by DEAE-TOYOPEARL chromatography and electrical elution by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by reverse-phase chromatography. The D/L ratio of aspartic acid in the protein isolated was 0.12. The molecular weight of this protein was estimated to be 22,500 by SDS-PAGE. The high content of serine, glycine and glutamic acid was noteworthy. It has been considered that the presence of DAsp in the living body is caused by racemization closely related to aging. The age of bovines used was relatively young (5 years old). If the racemization was caused by aging, the presence of DAsp in the relatively young bovine lens suggested that the aging of the lens protein may start at a relatively young age. The protein containing DAsp may be generally present in lens beyond species such as mouse, bovine and human.     

Acid-Catalyzed nucleophilic substitution and racemization of 3-methoxy-N-desmethyldiazepam enantiomers in methanol

pK_a1 values of 3-methoxy-N-desmethyldiazepam in acetonitrile and methanol containing various acid concentrations were determined by spectrophotometry to be 3.5 and 1.3, respectively. Temperature-dependent racemization of enantiomeric 3-methoxy-N-desmethyldiazepam in methanol containing 0.5 M H₂SO₄ was studied by circular dichroism spectropolorimetry and the racemization reactions were found to follow apparent first-order kinetics. Thermodynamic parameters of the racemization reaction were found to be: E_act = 18.8 kcal/mol, and at 25°C: ΔH^? = 18.3 kcal/mol, ΔS^? = ?14.8 entropy unit, and ΔG^? = 22.7 kcal/mol, respectively. The racemization had an isotope effect (k_H/k_D) of 1.6 at 42°C. Based on the results of this report and those of earlier reports by other investigators, a nucleophilically solvated C3 carbocation intermediate resulting from either a P (plus) or an M (minus) conformation is proposed to be an intermediate and responsible for the stereoselective nucleophilic substitution and the subsequent racemization of 3-methoxy-N-desmethyldiazepam enantiomers. © 1993 Wiley-Liss, Inc.     

The Stability of Some Selected Amino Acids Under Attempted Redox Constrained Hydrothermal Conditions

In order to evaluate the stability of aspartic acid, serine, leucine, and alanine under redox buffered hydrothermal conditions, a series of experiments have been performed. Thepyrite-pyrrhotite-magnetite (PPM) mineral assemblage was used in the experimental systems in order to constrain the oxygen fugacity. Likewise, the K-feldspar-muscovite-quartz (KMQ) assemblage was added to control the hydrogen ion activity during the experiments. The purpose was to compare the relative stabilities in buffered and unbuffered experiments.The experiments were conducted at 200 °C and 50 bar in Teflon coated autoclaves. Glycine, which wasnot present initially, started to appear at an earlystage in the experimental systems and is believed tobe the result of decomposition of serine. Similarly,the increase in relative abundance of alanine is likely to be the result of decomposition of serine. Decomposition rates of leucine, alanine and aspartic acid were found to be lower in experiments containing the redox buffer assemblagepyrite-pyrrhotite-magnetite than in non-redox bufferedexperiments. The decomposition rate of serine washigher in buffered experiments, which indicates thata transformation pathway via dehydration of serine todehydroalanine followed by reduction to alanine ispromoted by reducing conditions.     

Native chemical ligation in dimethylformamide can be performed chemoselectively without racemization

Native chemical ligation of unprotected peptides in organic solvents has been previously reported as a fast, efficient, and suitable method for coupling of hydrophobic peptides. However, it has not been determined whether the reaction can be carried out without possible side reactions or racemization. Here, we present a study on the chemoselectivity of this method by model reactions designed to test the reactivity of Arg and Lys side chains as well as that of α‐amino groups. A possible racemization of the C‐terminal amino acid of the N‐terminal peptide was also investigated. The results show that ligation in organic solvents can be conducted chemoselectively without side reactions with other nucleophilic groups. Furthermore, no racemization of the C‐terminal amino acid was observed if both educts were added simultaneously. Thus, native chemical ligation can be performed either in aqueous buffer systems or in organic solvents paving the way for the synthesis of larger hydrophobic peptides and/or membrane proteins. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.