The racemization of free and peptide-bound serine and aspartic acid at 100°C as a function of pH: Implications for in vivo racemization

The relative racemization rates of free and peptide-bound serine and aspartic acid have been studied as a function of pH at 100°C. These results have been used to explain the observed relative in vivo racemization rates of serine and aspartic acid in human tooth dentin and human ocular lens proteins.     

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.     

Racemization of Amino Acid Residues in Casein Roasted with Glucose and/or Methyl Linoleate

As model systems of foods, casein mixed with glucose and/or methyl linoleate were heated in an electric roaster at 120~230°C for 20 min under air or nitrogen and racemization of amino acid residues in these roasted materials was investigated by capillary column gas chromatography. On roasting the food model systems as well as in the case of pure protein, racemization in the amino acid residues occurred, and aspartic acid, glutamic acid and alanine residues were remarkably racemized. Correlation between the remaining ratio and the degree of racemization of amino acids is observed. It has been found that some components of food model systems, such as reducing sugar and lipid, promote the decomposition and racemization of amino acid residues.     

北京猿人和丁村人的氨基酸年龄测定

本文试图用北京猿人和山西丁村人所在地层中伴生动物化石内的天门冬氨酸、异亮氨酸外消旋程度推算北京猿人和山西丁村人地点的年龄和山顶洞所经历的平均温度。     

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.     

Geochemical evolution of amino acids in dentine of Pleistocene bears

A linear correlation was established between aspartic acid racemization ratio from cave bear dentine collagen and absolute dating. The high correlation coefficient obtained allowed age calculation through amino acid racemization. Aspartic acid and glutamic acid racemization kinetics have also been explored in dentine from a North American black bear (Ursus americanus Pallas). Three sample sets were prepared for kinetic heating experiments in nitrogen atmosphere: one water soaked, one with a water-saturated nitrogen atmosphere, and one without any moisture. It was possible to show that the presence of water is a factor controlling amino acid racemization rate. The aspartic acid in a heating experiment at 105 degrees C shows an "apparent kinetics reversal" which can be explained by a progressive hydrolysis of amino acid chains (proteins and polypeptides). Because of the low potential of collagen preservation over long periods of time, the apparent kinetics reversal phenomenon will not affect the dating of old material where no traces of collagen remain. An apparent kinetics reversal was not observed in glutamic acid, which racemizates more slowly.     

Age-related accumulation of the advanced glycation endproduct pentosidine in human articular cartilage aggrecan: the use of pentosidine levels as a quantitative measure of protein turnover.

During aging, non-enzymatic glycation results in the formation and accumulation of the advanced glycation endproduct pentosidine in long-lived proteins, such as articular cartilage collagen. In the present study, we investigated whether pentosidine accumulation also occurs in cartilage aggrecan. Furthermore, pentosidine levels in aggrecan subfractions of different residence time were used to explore pentosidine levels as a quantitative measure of aggrecan turnover. In order to compare protein turnover rates, protein residence time was measured as racemization of aspartic acid. As has previously been shown for collagen, pentosidine levels increase with age in cartilage aggrecan. Consistent with the faster turnover of aggrecan compared to collagen, the rate of pentosidine accumulation was threefold lower in aggrecan than in collagen. In the subfractions of aggrecan, pentosidine levels increased with protein residence time. These pentosidine levels were used to estimate the half-life of the globular hyaluronan-binding domain of aggrecan to be 19.5 years. This value is in good agreement with the half-life of 23.5 years that was estimated based on aspartic acid racemization. In aggrecan from osteoarthritic (OA) cartilage, decreased pentosidine levels were found compared with normal cartilage, which reflects increased aggrecan turnover during the OA disease process. In conclusion, we showed that pentosidine accumulates with age in aggrecan and that pentosidine levels can be used as a measure of turnover of long-lived proteins, both during normal aging and during disease.     

DNA decay rate in papyri and human remains from Egyptian archaeological sites

The writing sheets made with strips from the stem (caulis) of papyri (Cyperus papyrus) are one of the most ingenious products of ancient technology. We extracted DNA from samples of modern papyri varying in age from 0-100 years BP and from ancient specimens from Egypt, with an age-span from 1,300-3,200 years BP. The copy number of the plant chloroplast DNA in the sheets was determined using a competitive PCR system designed on the basis of a short (90 bp) tract of the chloroplast's ribulose bisphosphate carboxylase large subunit (rbcL) gene sequence. The results allowed us to establish that the DNA half-life in papyri is about 19-24 years. This means that the last DNA fragments will vanish within no more than 532-672 years from the sheets being manufactured. In a parallel investigation, we checked the archaeological specimens for the presence of residual DNA and determined the extent of racemization of aspartic (Asp) acid in both modern and ancient specimens, as a previous report (Poinar et al. [1996], Science 272:864-866) showed that racemization of aspartic acid and DNA decay are linked. The results confirmed the complete loss of authentic DNA, even in the less ancient (8th century AD) papyri. On the other hand, when the regression for Asp racemization rates in papyri was compared with that for human and animal remains from Egyptian archaeological sites, it proved, quite surprisingly, that the regressions are virtually identical. Our study provides an indirect argument against the reliability of claims about the recovery of authentic DNA from Egyptian mummies and bone remains.     

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.     

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.     

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.     

Racemization of Individual Aspartate Residues in Human Myelin Basic Protein

Human myelin basic protein (MBP), a long-lived brain protein, undergoes gradual racemization of its amino acids, primarily aspartic acid and serine. Purified protein was treated at neutral pH with trypsin to yield peptides that were separated by HPLC using a C18 column. Twenty-nine peptides were isolated and analyzed for amino acid composition and aspartate racemization. Each aspartate and asparagine in the protein was racemized to a different extent, ranging from 2.2 to 17.1% D isomer. When the racemization was examined in terms of the beta-structure model of MBP, a correlation was observed in which six aspartate/asparagine residues assumed to be associated with myelin membrane lipids showed little racemization (2.2-4.9% D isomer), whereas five other aspartate residues were more highly racemized (9.9-17.1% D isomer). Although the observed aspartate racemization may be related to steric hindrance by neighboring residues and/or the protein secondary structure, interaction of aspartates with membrane lipids may also be a major factor. The data are compatible with a model in which each MBP molecule interacts with adjacent cytoplasmic layers of myelin membrane through a beta-sheet on one surface and loops and helices on the other surface, thereby stabilizing the myelin multilamellar structure.     

Propensity for spontaneous succinimide formation from aspartyl and asparaginyl residues in cellular proteins

One mechanism for the spontaneous degradation of polypeptides is the intramolecular attack of the peptide bond nitrogen on the side chain carbonyl carbon atom of aspartic acid and asparagine residues. This reaction results in the formation of succinimide derivatives and has been shown to be largely responsible for the racemization, isomerization, and deamidation of these residues in several peptides under physiological conditions (Geiger, T. & Clarke, S. J. Biol. Chem. 262, 785-794 (1987]. To determine if similar reactions might occur in proteins, I examined the sequence and conformation about aspartic acid and asparagine residues in a sample of stable, well-characterized proteins. There did not appear to be any large bias against dipeptide sequences that readily form succinimides in small peptides. However, it was found that aspartyl and asparaginyl residues generally exist in native proteins in conformations where the peptide bond nitrogen atom cannot approach the side chain carbonyl carbon to form a succinimide ring. These orientations also represent energy minimum states, and it appears that this factor may account for a low rate of spontaneous damage to proteins by succinimide-linked reactions. The presence of aspartic acid and asparagine residues in other conformations, such as those in partially denatured, conformationally flexible regions, may lead to more rapid succinimide formation and contribute to the degradation of the molecule. The possible role of isoimide intermediates, formed by the attack of the peptide oxygen atom on the side chain carboxyl group, in protein racemization, isomerization, and deamidation is also considered.     

Chromatography as a means of selecting effective strains ofRhizobia

Summary 1. In 0.1 ml of bleeding sap of uninoculated plants aspartic acid was found to occur as the sole amino-acid usually in a small concentration. With larger quantities of sap we also found a spot in the chromatograms and in the electropherogram, that most probably is identical with threonine.2. Under the same conditions with a bleeding period of 4–7 hrs. aspartic acid was the only amino-acid detectable in the chromatogram of the bleeding sap of plants inoculated with ineffective strains.3. Sap of plants inoculated with effective strains contains aspartic acid asparagine, glutamine, hydroxyproline and threonine. Sometimes in addition to these substances 1 or 2 unknown substances were found, havingR _f-phenol values about identical with those of valine and leucine.4. Glutamic acid never was detected in the bleeding sap. We suggest, that glutamic acid formed in the nodules might be rapidly transformed into glutamine and transported in this form.5. The uptake of water is significantly higher with effective strains than with ineffective ones or with plants that have not been inoculated.6. With this technique it is possible to select effective strains within 3 weeks after planting.     

Abiotic Racemization Kinetics of Amino Acids in Marine Sediments

The ratios of d- versus l-amino acids can be used to infer the sources and composition of sedimentary organic matter. Such inferences, however, rely on knowing the rates at which amino acids in sedimentary organic matter racemize abiotically between the d- and the l-forms. Based on a heating experiment, we report kinetic parameters for racemization of aspartic acid, glutamic acid, serine, and alanine in bulk sediment from Aarhus Bay, Denmark, taken from the surface, 30 cm, and 340 cm depth below seafloor. Extrapolation to a typical cold deep sea sediment temperature of 3°C suggests racemization rate constants of 0.50×10⁻⁵–11×10⁻⁵ yr⁻¹. These results can be used in conjunction with measurements of sediment age to predict the ratio of d:l amino acids due solely to abiotic racemization of the source material, deviations from which can indicate the abundance and turnover of active microbial populations.     

The effect of humus substances on the level of free amino acids in wheat plants

The presence of potassium humate (commercial sample of Humussäure Riedel-de Haën A. G., Seelze-Hannover) in distilled water led to an increase in the content of alanine, aspartic and glutamic acid in the overground parts of wheat plants as compared with the content of these substances in control plants grown in distilled water. In plants cultivated on nutrient solutions a higher level of alanine and glutamic acid and a considerably lower level of amides was found than in plants grown in distilled water and in water with humate. Generally the content of amino acids was higher in the overground parts than in the roots after a cultivation period of one week as well as of 14 day. In the overground parts of 1 days old plants the level of aspartic acid, asparagine and alanine was found to be higher and that of glutamine lower than in seven days old plants. In the roots of the examined plants a decrease of the amino acid content accurred almost in all cases after a cultivation period of 14 days as compared to one of 7 days.     

The influence of black bean aphid, Aphis fabae Scop., and its honeydew on the photosynthesis of sugar beet

The effect of black bean aphids on the photosynthesis of sugar beet plants was studied under glasshouse and field conditions. The presence of up to several hundred aphids per leaf had no significant effect on CO₂ exchange rates over a range of light intensities between complete darkness and light saturation. Artificially prepared honeydew, sprayed onto leaves in the same amounts and composition as was found on severely aphid-infested plants, covered 30% of the stomata on the upper epidermis but did not significantly alter the rate of photosynthesis of these leaves in the light or the rate of respiration in the dark. The stomata on the lower epidermis were uncovered and functional. High pressure liquid chromatography of aphid-produced honeydew detected 20 different amino-acids. Three amino-acids, aspartic acid, glutamic acid and gluta-mine, made up the bulk of the amino-acid weight in the honeydew produced on young plants, up till the 8 leaf-stage. In the 10 to 12 leaf-stage, several different amino-acids occurred in substantial amounts. The amino-acids to sugars ratio of the honeydew produced by the aphids decreased strongly as the sugar beet plants aged: from 1:6 in plants with 3 or 4 leaves to 1:25 in plants having 10 to 12 leaves.     

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.     

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.     

The effect of nitrogen source on levels of amino acids in peas

Summary When nitrogen fixation in peas was partially replaced by nitrate assimilation as the source of nitrogen, an increase was found in the amount of soluble nitrogen that could be extracted from the fruits of the plants, and within this soluble fraction, increases were found in the levels of some of the acidic amino acids. Levels of protein amino acids in the peas were generally unaffected by the type of nitrogen source except for the level of aspartic acid which was about 20 per cent lower in peas supplied with nitrate. No differences were found in the proportions of the essential amino acids.