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Detailed investigations of the reaction pathway to trinuclear oxo-bridged uranyl complexes.

High pressure processing (HPP) represents a non-thermal preservation technique for the gentle treatment of food products. Information about the impact of HPP on lipophilic food ingredients (e.g., carotenoids, vitamin E) is still limited in more complex matrices such as kale. Both the variation of pressure levels (200–600 MPa) and different holding times (5–40 min) served as HPP parameters. Whereas a slightly decreasing solvent extractability mostly correlated with increasing pressure regimes; the extension of holding times resulted in elevated extract concentrations, particularly at high-pressures up to 600 MPa. Surprisingly, slightly increasing bioaccessibility correlated with both elevated pressures and extended holding times, indicating matrix-dependent processes during in vitro digestion, compared to results of extractability. Moreover, the verification of syringe filters for digest filtration resulted in the highest relative recoveries using cellulose acetate and polyvinylidene difluoride membranes. The α-tocopherol equivalent antioxidant capacity (αTEAC) and oxygen radical antioxidant capacity (ORAC) assays of treated kale samples, chopped larger in size, showed increased antioxidant capacities, regarding elevated pressures and extended holding times. Consequently, one may conclude that HPP was confirmed as a gentle treatment technique for lipophilic micronutrients in kale. Nevertheless, it was indicated that sample pre-treatments could affect HP-related processes in food matrices prior to and possibly after HPP.

Background: Serotonin has been implicated in impulsive behaviours such as temporal discounting. While animal studies and theoretical approaches suggest that reduced tonic serotonin levels increase temporal discounting rates and vice versa, evidence from human studies is scarce and inconclusive. Furthermore, an important modulator of serotonin signalling, a genetic variation in the promoter region of the serotonin transporter gene ( 5-HTTLPR), has not been investigated for temporal discounting so far. Objective: First, the purpose of this study was to test for a significant association between 5-HTTLPR and temporal discounting. Second, we wished to investigate the effect of high/low tonic serotonin levels on intertemporal choice and blood oxygen-level-dependent response, controlling for 5-HTTLPR. Methods: We tested the association of 5-HTTLPR with temporal discounting rates using an intertemporal choice task in 611 individuals. We then manipulated tonic serotonin levels with acute tryptophan interventions (depletion, loading, balanced) in a subsample of 45 short (S)-allele and 45 long (L)/L-allele carriers in a randomised double-blind crossover design using functional magnetic resonance imaging and an intertemporal choice task. Results: Overall, we did not find any effect of serotonin and 5-HTTLPR on temporal discounting rates or the brain networks associated with valuation and cognitive control. Conclusion: Our findings indicate that serotonin may not be directly involved in choices including delays on longer timescales such as days, weeks or months. We speculate that serotonin plays a stronger role in dynamic intertemporal choice tasks where the delays are on a timescale of seconds and hence are therefore directly experienced during the experiment.

The last two decades saw a steady increase of high hydrostatic pressure (HHP) used for treatment of foods. Although the science of biomaterials exposed to high pressure started more than a century ago, there still seem to be a number of unanswered questions regarding safety of foods processed using HHP. This review gives an overview on historical development and fundamental aspects of HHP, as well as on potential risks associated with HHP food applications based on available literature. Beside the combination of pressure and temperature, as major factors impacting inactivation of vegetative bacterial cells, bacterial endospores, viruses, and parasites, factors, such as food matrix, water content, presence of dissolved substances, and pH value, also have significant influence on their inactivation by pressure. As a result, pressure treatment of foods should be considered for specific food groups and in accordance with their specific chemical and physical properties. The pressure necessary for inactivation of viruses is in many instances slightly lower than that for vegetative bacterial cells; however, data for food relevant human virus types are missing due to the lack of methods for determining their infectivity. Parasites can be inactivated by comparatively lower pressure than vegetative bacterial cells. The degrees to which chemical reactions progress under pressure treatments are different to those of conventional thermal processes, for example, HHP leads to lower amounts of acrylamide and furan. Additionally, the formation of new unknown or unexpected substances has not yet been observed. To date, no safety-relevant chemical changes have been described for foods treated by HHP. Based on existing sensitization to non-HHP-treated food, the allergenic potential of HHP-treated food is more likely to be equivalent to untreated food. Initial findings on changes in packaging materials under HHP have not yet been adequately supported by scientific data.

High pressure processing (HPP) is a novel food processing technology that uses minimal heat and preservatives. It can inactivate a variety of pathogenic and spoilage bacteria to ensure safe foods and extend shelf life by maintaining nutritional and sensory attributes. The present investigation studied the effects of HPP (200, 400, 600 MPa; 5, 10 min; room temperature) on the following parameters: contents of carotenoids and vitamin E and antioxidant capacity (AOC) in spinach and rosehip puree; chlorophyll contents in spinach and in vitro bioaccessibility of carotenoids in rosehip puree. Contents of carotenoids, chlorophylls and vitamin E were determined by HPLC. AOC was analysed by several assays (total phenolics, TEAC, ORAC). In spinach, HPP led to a significant increase of the content of carotenoids, chlorophylls and vitamin E, probably due to an increased extractability. Total carotenoid content in rosehip puree was almost unchanged by HPP with single carotenoids behaving differently. The in vitro digestion model showed that 42.2 ± 7.4% of the total carotenoids of the rosehip puree was released and is potentially available for absorption. HPP had no significant effect on the bioaccessibility of total carotenoids. No distinct trend for the effects of HPP on the tocopherol contents in both food samples could be deduced. Mostly, no changes were induced by pressure. In both plant foods, the AOC was retained. The results provide further scientific evidence of the benefits of HPP in retaining micronutrients and phytochemical properties of plant foods.

To investigate the influence of high hydrostatic pressure on the reactions of glyoxal with lysine residues, the formation of end-products, namely N-e-(carboxymethyl)lysine (CML) and 1,3-bis(5-amino-5-carboxypentyl)imidazolium salt (GOLD), was studied in model systems via LC–MS. Following a kinetic interpretation, reaction volumes were calculated. Corresponding values of +5.4 (CML) and +9.9 cm³/mol (GOLD) indicate a suppressive effect of pressure on the formation of these glycation compounds. Above this, several other known end-products, namely N(6)-glycoloyllysine (GALA), N(6)-[2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl]lysine (GOLA), and reaction intermediates, were qualitatively monitored and proofed a similar behavior compared to the GOLD formation pathway. The reasons for the influence of pressure are discussed in the context of the reaction mechanism.

Serotonin has been implicated in impulsive behaviours such as temporal discounting. While animal studies and theoretical approaches suggest that reduced tonic serotonin levels increase temporal discounting rates and vice versa, evidence from human studies is scarce and inconclusive. Furthermore, an important modulator of serotonin signalling, a genetic variation in the promoter region of the serotonin transporter gene (First, the purpose of this study was to test for a significant association betweenWe tested the association ofOverall, we did not find any effect of serotonin andOur findings indicate that serotonin may not be directly involved in choices including delays on longer timescales such as days, weeks or months. We speculate that serotonin plays a stronger role in dynamic intertemporal choice tasks where the delays are on a timescale of seconds and hence are therefore directly experienced during the experiment.

Two novel 46-membered Co(II) metallomacrocycles, [Co2Cl4(L1)2]·CH2Cl2 and [Co2(NO3)2(L1)2(H2O)2⊂(NO3)](NO3)·4H2O, with 1,4-bis[4-(2-pyridylmethylideneamino)phenyloxy]benzene (L1) have been synthesized and characterized. Both Co(II) centers in these dinuclear complexes possess an octahedral coordination environment formed by a N4 donor set of two L1 ligands and two chloride or one monodentate nitrate and one water molecule, respectively. Whereas in the first case disordered solvent molecules are present in the macrocycle's cavity in the second one a nitrate anion is embedded. Furthermore, the molecular structures of L1, L3 and L4 were determined and solution studies of the complex formation of these ligands with Co(II), Ni(II) and Cu(II) in dichloromethane/methanol (v/v = 1:1) using UV/Vis and ESI-MS spectroscopic measurements were performed. In contrast to the self-assembling behavior of the bis(2-pyridylimine) ligand L1 the bis(3-pyridylimine) ligand L4 was also demonstrated to form metallogels when reacted with NiCl2·6H2O in tetrahydrofuran under defined conditions.

Serotonin (5-HT) plays a key role in different aspects of value-based decision-making. A recent framework proposed that tonic 5-HT (together with dopamine, DA) codes future average reward expectations, providing a baseline against which possible choice outcomes are compared to guide decision-making. To test whether high 5-HT levels decrease loss aversion, risk-seeking for gains, and risk-seeking for losses. In a first session, 611 participants were genotyped for 5-HTTLPR and performed a mixed gambles (MGA) task and two probability discounting tasks for gains and losses, respectively (PDG/PDL). Afterwards, a subsample of 105 participants (44 with S/S, 6 with S/L, 55 with L/L genotype) completed the pharmacological study using a crossover design with tryptophan depletion (ATD), loading (ATL), and balanced (BAL) conditions. The same decision constructs were assessed. We found increased risk-seeking for losses in S/S compared to L/L individuals at the first visit (p = 0.002). Neither tryptophan depletion nor loading affected decision-making, nor did we observe an interaction between intervention and 5-HTTLPR genotype. Our data do not support the idea that transient changes of tonic 5-HT affect value-based decision-making. We provide evidence for an association of 5-HTTLPR with risk-seeking for losses, independent of acute 5-HT levels. This indicates that the association of 5-HTTLPR and risk-seeking for losses is mediated via other mechanisms, possibly by differences in the structural development of neural circuits of the 5-HT system during early life phases.

Whey protein was shown to reduce blood glucose responses in humans and various other positive effects have been attributed to this protein. In contrast, studies using glycomacropeptide (GMP) as part of the whey fraction of bovine milk are rare. We, therefore, studied the postprandial responses to GMP administration in humans with impaired glucose tolerance compared to the effects of pure whey protein in a random design. Fifteen prediabetic volunteers received on different occasions one of three test drinks containing 50 g of maltodextrin19 (MD19) alone or in combination with either 50 g GMP or 50 g whey protein isolate (WPI). Blood was collected over 4 h with analysis of blood glucose and hormones, gastric emptying rate as well as plasma amino- and fatty acids, β-hydroxybutyrate and acylcarnitines. The WPI drink reduced the AUC of venous blood glucose compared to the MD19 drink in the prediabetic group by 11% (p = 0.0018) whereas GMP reduced the AUC by 18% (p

Acid casein in phosphate buffer was used as a simplified substrate for cross-linking experiments with microbial transglutaminase. The importance of the intensity of enzyme treatment on the rheology of acid gels was evaluated by preparing mixtures of casein that were cross-linked to a different extent and a native reference to obtain samples with a similar polymerisation degree. The stiffness of acid gels from caseins incubated longer then 3 h was always lower at identical polymerisation degree larger than approximately 30%. The quantification of isopeptides formed by the enzyme reveals a substantial increase of bonds at a stage of cross-linking where only 10% of monomers are available. This indicates that cross-linking must occur between and/or within already existing aggregates, which become larger and less flexible, and supports the hypothesis that, starting with a critical aggregate size the network formation is somewhat impaired by the size and reduced flexibility of the proteins.

Processing of food modifies nutrient contents and food matrices. The present analyses provide information about the effects of heat steam sterilization (HSS), high pressure processing (HPP) and common household cooking on the stability of carotenoids and chlorophylls in spinach, parsley, dill and kale. For HSS, samples were heated in an autoclave for 5, 10, 15 and 20 min at 121 °C. Additionally, one sample was treated via HPP at 200, 400 and 600 MPa for 5, 10 and 40 min at room temperature. Carotenoid and chlorophyll contents were determined by reversed-phase HPLC. Lutein was the most abundant carotenoid in all samples. Furthermore, violaxanthin, zeaxanthin, β-carotene, chlorophylls a and b were quantified. Thermal treatment via HSS led to a significant decline of contents of lutein, chlorophylls a and b. HPP did not reduce the amounts of xanthophylls and chlorophylls. Conclusively, HPP seems to be a good alternative to HSS for the preservation of xanthophyll-rich food.

Pathological alterations in cell functions are frequently accompanied by metabolic reprogramming including modifications in amino acid metabolism. Amino acid detection is thus integral to the diagnosis of many hereditary metabolic diseases. The development of malignant diseases as metabolic disorders comes along with a complex dysregulation of genetic and epigenetic factors affecting metabolic enzymes. Cancer cells might transiently or permanently become auxotrophic for non-essential or semi-essential amino acids such as asparagine or arginine. Also, transformed cells are often more susceptible to local shortage of essential amino acids such as methionine than normal tissues. This offers new points of attacking unique metabolic features in cancer cells. To better understand these processes, highly sensitive methods for amino acid detection and quantification are required. Our review summarizes the main methodologies for amino acid detection with a particular focus on applications in biomedicine and cancer, provides a historical overview of the methodological pre-requisites in amino acid analytics. We compare classical and modern approaches such as the combination of gas chromatography and liquid chromatography with mass spectrometry (GC-MS/LC-MS). The latter is increasingly applied in clinical routine. We therefore illustrate an LC-MS workflow for analyzing arginine and methionine as well as their precursors and analogs in biological material. Pitfalls during protocol development are discussed, but LC-MS emerges as a reliable and sensitive tool for the detection of amino acids in biological matrices. Quantification is challenging, but of particular interest in cancer research as targeting arginine and methionine turnover in cancer cells represent novel treatment strategies.

Using LC-MS/MS and isotopically labeled standard substances, quantitation of free Maillard reaction products (MRPs), namely, N(ε)-(carboxymethyl)lysine (CML), 5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (pyrraline, PYR), N(δ)-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H), and N(ε)-fructosyllysine (FL), in bovine milk was achieved. Considerable variations in the amounts of the individual MRPs were found, most likely as a consequence of the nutritional uptake of glycated proteins. When comparing commercial milk samples labeled as originating from "organic" or "conventional" farming, respectively, significant differences in the content of free PYR (organic milk, 20-300 pmol/mL; conventional milk, 400-1000 pmol/mL) were observed. An analysis of feed samples indicated that rapeseed and sugar beet are the main sources for MRPs in conventional farming. Furthermore, milk of different dairy animals (cow, buffalo, donkey, goat, ewe, mare, camel) as well as for the first time human milk was analyzed for free MRPs. The distribution of their concentrations, with FL and PYR as the most abundant in human milk and with a high individual variability, also points to a nutritional influence. As the components of concentrated feed do not belong to the natural food sources of ruminants and equidae, free MRPs in milk might serve as indicators for an adequate animal feeding in near-natural farming and can be suitable parameters to distinguish between an "organic" and "conventional" production method of milk.

High-pressure treatment, e.g. used as an alternative method for food preservation, affects protein cross-linking and glycation reactions. These reactions were monitored by using mainly milk proteins in the absence and presence of different saccharides or dicarbonyl compounds. Without carbohydrates, protein cross-linking of casein is enhanced by pressure through the formation of dehydroalanine-derived lysinoalanine. A similar effect can be observed in wool, where pressure accelerates the formation of lanthionine. In contrast, saccharide or dicarbonyl compound-induced cross-linking is constrained by high pressures. Despite this, pressure is able to accelerate the degradation of sugar or carbonyl compounds, as could be seen by the measurement of their residual contents in the examined test preparations.

After incubation of hen egg white lysozyme (HEWL) with microbial transglutaminase (mTG) under high pressure (400-600 MPa for 30 min at 40 °C), the formation of HEWL oligomers was observed via SDS electrophoresis. At atmospheric pressure, HEWL represents no substrate for mTG. Likewise, enzymatic treatment following a pretreatment with high pressure did not lead to oligomerization. Reactive amino acid side chains were identified by peptide mapping after tryptic digestion using RP-HPLC with ESI-TOF-MS. Isopeptide-containing peptide fragments were found only in HEWL samples simultaneously treated with enzyme and pressure. It was found that mTG exclusively cross-links HEWL under high pressure by formation of an isopeptide between lysine at position 1 and glutamine at position 121 in the peptide chain. Therefore, a pressure-induced partial and reversible unfolding of the protein with exposure of lysine and glutamine side chains has to occur, resulting in a site-directed oligomerization of HEWL by mTG. The enzymatic modification of HEWL by mTG under high pressure offers interesting perspectives for further functionalization reactions.

African yam bean ( Sphenostylis stenocarpa ) starch was subjected to heat moisture treatments at 18% (HMT-18), 21% (HMT-21), 24% (HMT-24), 27% (HMT-27) and excess (Annealing) moisture levels. Proximate chemical composition of the starch samples revealed that the moisture content of the starches ranged between 6.7% and 12.5%. Following modification of the native starch, there was a reduction in the moisture level of the heat moisture treated starches from HMT-18 to HMT-27. However, the annealed starch (HMT-ANN) retained higher moisture content compared to native starch (AYB-Native). The carbohydrate, protein, ash, amylose and fat content reduced with all the forms of heat treatments. At the temperature range studied (60–90 °C), increasing level of heat moisture treatments reduced the solubility and swelling capacity. pH also exert a profound effect both on the solubility and the swelling of the starch. Increasing degree of alkalinity increased both solubility and swelling capacity. In the native and modified starch samples, replacement of the wheat flour by the starch resulted in increased alkaline water retention of the blends. Water absorption capacity of the starch increased with the severity of moisture treatments, while the oil absorption capacity decreased. Apart from HMT-18, there was improved gel forming capacity of all the other heat-modified starches. Pasting temperature increased after hydrothermal modifications, whereas peak viscosity (P v ), Hot Paste Viscosity (H v ), setback and breakdown values all reduced after heat moisture treatments. All the starches were of type-B viscosity. Differential scanning calorimetry studies revealed that heat moisture treatment shifted the onset temperature (T o ), peak temperature(T p ) and conclusion temperature (T c ) to higher values. The gelatinisation temperature of the annealed starch was comparable to native starch. In addition, gelatinisation band of the native starch increased progressively from HMT-18 to HMT-27. Heat moisture treatment reduced the gelatinsation enthalpy (ΔH), while the enthalpy of retrogradation(ΔH r ) increased with the storage time of the gelatinised starch. Retrogradation enthalpy of the heat moisture conditioned starches were lower than the value obtained for the native starch. X-ray diffraction studies of the starch indicated that all the starch samples showed the type-C diffraction pattern. Differences were however observed in their degree of crystallinity. Native starch exhibited the lowest crystallinity (20%) while annealed starch had the highest crystallinity (27%) Microscopy studies revealed surface indentation, formation of groves in the central region, folding of starch granules and formation of doughnut-like appearance in some of the starch samples.

Kinetics for the reaction of microbial transglutaminase (MTG) with individual caseins in a TRIS-acetate buffer at pH 6.0 was evaluated under atmospheric pressure (0.1 MPa) and high pressure (400 MPa) at 40 °C. The reaction was monitored under the following limitations: The kinetics from the initial velocities was obtained from nonprogressive enzymatic reactions assuming that the individual catalytic constants of reactive glutamine residues are represented by the reaction between MTG and casein monomers. Enzyme reaction kinetics carried out at 0.1 MPa at 40 °C showed Henri-Michaelis-Menten behavior with maximal velocities of 2.7 ± 0.02 × 10(-3), 0.8 ± 0.01 × 10(-3), and 1.3 ± 0.30 × 10(-3) mmol/L · min and K(m) values of 59 ± 2 × 10(-3), 64 ± 3 × 10(-3), and 50 ± 2 × 10(-3) mmol/L for β-, α(s1)-, and acid casein, respectively. Enzyme reaction kinetics of β-casein carried out at 400 MPa and 40 °C also showed a Henri-Michaelis-Menten behavior with a similar maximal velocity of 2.5 ± 0.33 × 10(-3) mmol/L · min, but, comparable to a competitive inhibition, the K(m) value increased to 144 ± 34 × 10(-3) mmol/L. The reaction of MTG with α(s1)-casein under high pressure did not fit in to Henri-Michaelis-Menten kinetics, indicating the complex influence of pressure on protein-enzyme interactions.

In the present work, we show for the first time, that N ɛ -carboxymethyllysine is the major product of the in vitro non-enzymatic glycation reaction between fibrillar collagen and glucuronic acid . Dual diffusion membrane system was effectively used for oriented crystal growth of octacalcium phosphate/hydroxyapatite on the biomimetically carboxymethylated collagen fibrils. We hypothesize that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions in such a way that a critical nucleus of ions can be formed, leading to the formation of the mineral under specific micro-environment conditions achieved by using diffusion membrane system.

Developing new biopolymer-based materials with bio-identical properties is a significant challenge in modern science. One interesting route to this goal involves the biomineralization of collagen, a pre-structured and widely available protein, into a material with interesting properties. A prerequisite for biomineralization is the ability of cations (e.g., calcium) to bind to the protein and to result in concert with appropriate anions (e.g., phosphate) in composite material with e.g., bone-like properties. In order to increase the number of binding sites it is necessary to modify the protein prior to mineralization. For this glucuronic acid (GA) was used due to its carbonyl and carboxyl groups to derivatize proteinogenic amino groups transferring them into negatively charged carboxyl groups. Our experiments showed for the first time, that Nepsilon-carboxymethyllysine is the major product of in vitro non-enzymatic glycosylation of collagen by glucuronic acid. For an unequivocal determination of the reaction products, the lysine residues of collagen and of the model peptide were carboxymethylated through a reductive alkylation with glyoxalic acid and compared to the glucuronic acid derivatives. Beside their identical mass spectra the common structure elements could be confirmed with FTIR. Thus, in the context of matrix engineering, by producing Nepsilon-carboxymethyllysine, glucuronic acid offers a convenient way of introducing additional stable acidic groups into protein matrices.

Mixtures of N alpha-hippurylarginin, N alpha-hippuryllysine, and glyoxal were incubated in the absence and presence of N alpha-acetylcysteine in order to assess the individual reactivity of these nucleophilic amino acid residues. The incubations were performed under atmospheric and high hydrostatic pressure (400 MPa), and, at the same time, beta-casein was reacted with glyoxal. The results showed that arginine is the main partner for glyoxal in the absence of cysteine, whereas a lysine derivatization was not apparent. In the presence of cysteine, however, arginine was almost completely protected from the reaction, whereas a noticeable formation of lysine derivatives, mainly carboxymethyllysine, was observed. Based on these findings, a reaction mechanism is proposed to explain the influence of cysteine on the reaction.

Specific determination of N-(2-furoylmethyl)valine (FM-Val) together with furosine in acid hydrolyzates of human hemoglobin of healthy volunteers (n = 6) and diabetic patients (n = 14) by means of reversed-phase HPLC with electrospray ionization-time-of-flight mass spectroscopy is reported. Whereas FM-Val is formed during acid hydrolysis of the N-terminal hemoglobin adduct N-fructosylvaline, furosine results from acid degradation of lysine residues glycated at the epsilon-amino group. Quantification was based on the use of synthesized isotopomers, namely N-[2-(13C6)furoylmethyl]valine and N-epsilon-[2-(13C6)furoylmethyl]lysine, thus enabling interference-free detection and calibration. Taking the conversion factors into account, the amount of N-terminally bound N-fructosylvaline in human hemoglobin was between 518 and 774 pmol/mg protein for healthy volunteers and between 586 and 1426 pmol/mg protein for diabetic patients. Derivatization at the side chain of peptide-bound lysine residues to N-epsilon-fructosyllysine was from 1156 to 1753 pmol/mg protein for healthy controls and from 1191 to 2409 pmol/mg protein for diabetics. For these patients, the amount of N-fructosylvaline showed good correlation with the values for HbA(1c). The significantly higher relative extent of glycation at the N terminus compared to side-chain glycation points to a specific and intraindividual capacity for enzymatic deglycation in human erythrocytes, which can be assessed using the proposed method.

The purpose of this study was to evaluate the effects of microbial transglutaminase on the rheological and mechanical properties of gels from a commercial UHT milk made by acidification with d-gluconic-acid-δ-lactone. Although further heating of UHT milk, primarily carried out to inactivate the enzyme, showed a significant impact on gel firmness, we observed no enzyme-related effects at small deformation. The impact of the additional thermal treatment was also evident in large deformation measurements. In these experiments, gels from enzyme-treated milk revealed much higher elasticity and rupture force. Prolonged incubation time or increased enzyme concentration resulted in lower gel firmness (small deformation), indicating a reduced number of total bonds as a result of the restriction of proper rearrangements during acidification. Large deformation measurements revealed a heat-induced amplification of this effect, i.e., lower rupture forces at almost constant elasticity levels. In gels from milk, which was not subjected to thermal enzyme inactivation, we observed a further increase in both parameters. This is presumably caused by a combination of residual enzyme activity and of additional substrate, liberated during acidification.

Casein solutions (5% w/v) were treated with microbial transglutaminase (MTG) and glucono-delta-lactone (GDL) under varying conditions in order to obtain gels. Storage modulus (G') and gelation time of the gels were measured by oscillation rheometry, while protein cross-linking was determined by gel permeation chromatography. The addition of only GDL to milk resulted in very weak gels, while MTG on its own was not able to create gel networks. Simultaneous action of both ingredients led to gels, the firmness of which was linearly related to the added amount of MTG, but passed through a maximum with rising GDL concentrations. Using chromatographical analysis, increasing G' values were interrelated with the formation of MTG-induced oligomers. The gelation time was directly proportional to the GDL concentration but not influenced by the addition of MTG within the studied range of concentration.

Enzyme activity and corresponding secondary structure, measured by circular dichroism was analysed before und after treatment of microbial transglutaminase at different temperatures (40, 80°C) and pressures (0.1, 200, 400, 600 MPa). Irreversible enzyme inactivation was achieved at 80°C after 2 minutes at atmospheric pressure. Enzyme inactivation at 0.1, 200, 400, 600 MPa and 40°C followed first order kinetics. Increasing pressure reduced MTG activity, nevertheless the enzyme showed a residual activity of 50% after 12 min at 600 MPa. The analysis of the native enzyme exhibited well-defined proportions between α-helix, β-strand, β-turn and unordered structures. In contrast to heating, high-pressure treatment only at high levels induced significant decrease in the α-helix content, whereas β-strand substructures remained unaltered in both cases. Based on the known crystal structure of MTG it can be concluded that the active centre of the enzyme itself, which is located in an expanded β-strand domain, is relatively stable and pressure-induced inactivation is caused by a degradation of α-helix elements with corresponding influence on the tertiary structure.

Control over crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues. Highly acidic macromolecules are postulated intermediates in tissue mineralization, because they sequester many calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. A prerequisite for biomineralization is the ability of cations like calcium to bind to proteins and to result in concert with appropriate anions like phosphates or carbonates in composite materials with bone-like properties. For this mineralization process the proteins have to be modified with respect to acidification. In this study we modified the protein collagen by carboxymethylation using glucuronic acid. Our experiments showed unambigously, that Ne-carboxymethyllysine is the major product of the in vitro nonenzymatic glycation reaction between glucuronic acid and collagen. We hypothesized that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions so that a critical nucleus of ions can be formed, leading to the formation of the mineral. Thus, the self-organization of HAP nanocrystals on and within collagen fibrils was intensified by carboxymethylation. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010

Specific determination of N-(2-furoylmethyl)valine (FM-Val) together with furosine in acid hydrolyzates of human hemoglobin of healthy volunteers (n = 6) and diabetic patients (n = 14) by means of reversed-phase HPLC with electrospray ionization-time-of-flight mass spectroscopy is reported. Whereas FM-Val is formed during acid hydrolysis of the N-terminal hemoglobin adduct N-fructosylvaline, furosine results from acid degradation of lysine residues glycated at the epsilon-amino group. Quantification was based on the use of synthesized isotopomers, namely N-[2-(13C6)furoylmethyl]valine and N-epsilon-[2-(13C6)furoylmethyl]lysine, thus enabling interference-free detection and calibration. Taking the conversion factors into account, the amount of N-terminally bound N-fructosylvaline in human hemoglobin was between 518 and 774 pmol/mg protein for healthy volunteers and between 586 and 1426 pmol/mg protein for diabetic patients. Derivatization at the side chain of peptide-bound lysine residues to N-epsilon-fructosyllysine was from 1156 to 1753 pmol/mg protein for healthy controls and from 1191 to 2409 pmol/mg protein for diabetics. For these patients, the amount of N-fructosylvaline showed good correlation with the values for HbA(1c). The significantly higher relative extent of glycation at the N terminus compared to side-chain glycation points to a specific and intraindividual capacity for enzymatic deglycation in human erythrocytes, which can be assessed using the proposed method.

Microbial transglutaminase (mTG) mediated modification of bovine beta-lactoglobulin (bLG) at ambient and high hydrostatic pressure was investigated in order to characterize preferred sites of the crosslinking reaction by identifying reactive glutamine residues. bLG was labeled with triglycine (GGG) by incubation with mTG at ambient pressure or at 400 MPa, respectively, and was subjected to an enzymatic digestion with trypsin. The resulting peptides were separated and those containing glutamine residues modified with GGG were unambiguously identified using RP-HPLC with ESI-TOF-MS. For bLG treated with mTG at ambient pressure for 1 h at 40 degrees C, no labeling was observed, thus confirming that the native protein is no substrate for mTG. After incubation of the protein with mTG at 400 MPa for 1 h at 40 degrees C, four out of nine glutamine residues, namely at positions 5, 13, 35, and 59 were identified as accessible for the mTG catalyzed reaction, indicating partial unfolding of bLG under pressure and exposure of previously unaccesible glutamine residues. Thus, only a limited number of glutamine residues were substrates for mTG, which points to a pronounced substrate specificity of mTG toward individual glutamine residues within a protein.

The influence of enzymatic crosslinking by microbial transglutaminase (mTG) on the stability of casein micelles of ultrahigh temperature (UHT)-treated milk in the presence of EDTA (0-0.45 mM) or ethanol (0-74 vol%) as well as under high hydrostatic pressures up to 400 MPa was investigated. Disintegration of micelles and changes in micelle size were monitored by the measurement of turbidity as well as by dynamic light scattering. The results show that the incubation of UHTtreated milk with mTG resulted in an improved micelle stability toward disintegration on addition of EDTA, ethanol, or pressure treatment. Intramicellar formed isopetides significantly enhanced the stability of casein micelles. It is supposed that net-like crosslinks are formed within the external region of the micelles and they adopt the stabilizing role of colloidal calcium phosphate within the micelles, thus making the micelles less contestable for disrupting influences.