Nondisulfide Crosslinks Research Articles

Empowering canonical biochemicals with cross-linked novelty: Recursions in applications of protein cross-links.

Diversity in the biochemical workhorses of the cell-that is, proteins-is achieved by the innumerable permutations offered primarily by the 20 canonical L-amino acids prevalent in all biological systems. Yet, proteins are known to additionally undergo unusual modifications for specialized functions. Of the various post-translational modifications known to occur in proteins, the recently identified non-disulfide cross-links are unique, residue-specific covalent modifications that confer additional structural stability and unique functional characteristics to these biomolecules. We review an exclusive class of amino acid cross-links encompassing aromatic and sulfur-containing side chains, which not only confer superior biochemical characteristics to the protein but also possess additional spectroscopic features that can be exploited as novel chromophores. Studies of their in vivo reaction mechanism have facilitated their specialized in vitro applications in hydrogels and protein anchoring in monolayer chips. Furthering the discovery of unique canonical cross-links through new chemical, structural, and bioinformatics tools will catalyze the development of protein-specific hyperstable nanostructures, superfoods, and biotherapeutics.

Orthogonal Crosslinking: A Strategy to Generate Novel Protein Topology and Function.

Compared to the disulfide bond, other naturally occurring intramolecular crosslinks have received little attention, presumably due to their rarity in the vast protein space. Here we presented examples of natural non-disulfide crosslinks, which we refer to as orthogonal crosslinks, emphasizing their effect on protein topology and function. We summarize recent efforts on expanding orthogonal crosslinks by using either the enzymes that catalyze protein circularization or the genetic code expansion strategy to add electrophilic amino acids site-specifically in proteins. The advantages and disadvantages of each method are discussed, along with their applications to generate novel protein topology and function. In particular, we highlight our recent work on spontaneous orthogonal crosslinking, in which a carbamate-based crosslink was generated in situ, and its applications in designing orthogonally crosslinked domain antibodies with their topology-mimicking bacterial adhesins.

In use stability of chemical anticancer drugs and biologics

Background. For hospital pharmacists and nurses it is critical to have real data about the stability of injectable drugs in practical situations: opened drug formulation, after the reconstitution of lyophilized production or after the dilution in various vehicles. Unfortunately, these data are seldom available. The manufacturer’s stability data are usually quoted as “stable for 24 hours at 4 °C” not for the true reasons, but considering the possible bacterial contamination or due to the fact that stability tests were only conducted during a very short period. This problem is of paramount importance for therapeutic proteins, mainly monoclonal antibodies, because the latter are very expensive.
 Objective. To define the in use stability of anticancer drugs.
 Materials and methods. Analysis of literature data on this topic; own study on diluted rituximab stability.
 Results and discussion. Biologic drugs have revolutionized the treatment and prevention of many debilitating and life-threatening diseases. Biologics are much bigger and have a much more complicated structure than classical drugs. Principal causes of biologics’ instability include temperature changes, dilution, absorption, oxidation, shaking and shearing etc. Protein instability can be divided into physical and chemical instability. The former includes aggregation, denaturation and absorption on surfaces, and the latter – desamidation, disulfide bonds breakage and formation, oxidation, hydrolysis, isomerization, non-disulfide cross-linking, and deglycosylation. Instability of anticancer drugs and biologicals can lead to their toxicity, immunogenicity and efficacy loss. Instability can be overcome by the centralization of pharmacy handling, but firstly it needs to be thoroughly studied with the help of modern methods. The own study has revealed that the diluted rituximab is stable up to 6 months in polyolefin bags stored at 4 or 22 °C. The preparations can be realized without any problems. There also no fear of instability in case of thermal excursions. Such findings if introduced in practice can save about 150,000 euros a year.
 Conclusions. 1. In-use analysis of proteins requires several methods. 2. Instabilities are divided into physical and chemical. 3. Aggregation is an initial response to any stress and must be completely eliminated.

Hotspots of age-related protein degradation: the importance of neighboring residues for the formation of non-disulfide crosslinks derived from cysteine.

Over time, the long-lived proteins that are present throughout the human body deteriorate. Typically, they become racemized, truncated, and covalently cross-linked. One reaction responsible for age-related protein cross-linking in the lens was elucidated recently and shown to involve spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine. Cys residues are another potential source of DHA, and evidence for this was found in many lens crystallins. In the human lens, some sites were more prone to forming non-disulfide covalent cross-links than others. Foremost among them was Cys5 in βA4 crystallin. The reason for this enhanced reactivity was investigated using peptides. Oxidation of Cys to cystine was a prerequisite for DHA formation, and DHA production was accelerated markedly by the presence of a Lys, one residue separated from Cys5. Modeling and direct investigation of the N-terminal sequence of βA4 crystallin, as well as a variety of homologous peptides, showed that the epsilon amino group of Lys can promote DHA production by nucleophilic attack on the alpha proton of cystine. Once a DHA residue was generated, it could form intermolecular cross-links with Lys and Cys. In the lens, the most abundant cross-link involved Cys5 of βA4 crystallin attached via a thioether bond to glutathione. These findings illustrate the potential of Cys and disulfide bonds to act as precursors for irreversible covalent cross-links and the role of nearby amino acids in creating 'hotpsots' for the spontaneous processes responsible for protein degradation in aged tissues.

Effects of rare sugar d-allulose on heat-induced gelation of surimi prepared from marine fish.

d-Allulose (Alu), the C3-epimer of d-fructose, is a non-caloric sweetener (0.39 kcal g-1 ) with a suppressive effect on postprandial blood glucose elevation. The aim of this study was to investigate the effects of Alu used as a sweetener and gel improver instead of sucrose on heat-induced gelation of surimi. The puncture test of a heat-induced surimi gel showed that with 50 g kg-1 Alu the gel had 15% and 6% higher gel strength than the corresponding gel with sucrose (Suc) and with sorbitol (Sor), respectively. In addition, Alu-gel had 26% and 25% higher water-holding capacity (WHC) than Suc- and Sor-gel. Heating of myofibrillar protein with Alu, unlike Suc and Sor, facilitated the formation of both disulfide and non-disulfide crosslinks that might be associated with the mechanical properties and WHC of Alu-gel. Alu improves the mechanical properties and WHC of the heat-induced surimi gel. Furthermore, Alu is low in calories compared with Suc (4.0 kcal g-1 ) and Sor (3.0 kcal g-1 ). Thus Alu will be an alternative of Suc or Sor for developing surimi-based products with health benefits. © 2017 Society of Chemical Industry.

Concepts and experimental protocols towards a molecular level understanding of the mechanical properties of glassy, cross-linked proteins: Application to wheat gluten bioplastics

Linking the mechanical performance of glassy polymeric materials to their molecular characteristics is a major challenge, even more so when proteinaceous raw materials are used with a complex cross-linking chemistry and composition dependent molecular interactions. Here, wheat gluten was separated into gliadin-rich and glutenin-rich fractions. Blends prepared thereof were compression molded (130–150°C, 5–28min) into glassy, brittle materials. Molecular characteristics as well as mechanical properties in bending and compression modes were determined and their mutual relations extensively discussed. The compression data were used to qualitatively predict the flexural strength values. Experimental outcomes deviated from the predictions possibly because of stress concentrating contaminants in glutenin-rich and molecular network interruptions in gliadin-rich materials. Depending on molding conditions and protein composition either only disulfide bonds or also non-disulfide cross-links were present. It is suggested that disulfide based cross-links contribute less to material toughness than other types of covalent cross-links.

Developing rigid gliadin based biocomposites with high mechanical performance

We aim to produce unidirectional fiber composites with high mechanical performance based on flax fibers and a rigid gliadin matrix. As a fraction from wheat gluten, gliadin is soluble in alcohol containing media. The fabrication process did not involve any further solvents or plasticizers. Finally, samples were cooled at different rates. Overall, the cooling rate does not strongly affect the mechanical properties although slowly cooled materials contain a higher amount of non-disulfide cross-links, next to disulfide bonds within the gliadin matrix. At 40% fiber volume fraction, flax/gliadin composites with a flexural modulus and strength of respectively 21.5GPa and 240MPa were obtained when loaded in the longitudinal direction. These high values demonstrate that in this composite fabrication process, a good impregnation of the polymer matrix in between the fiber bundles has been achieved. However, the fiber–matrix adhesion, as measured by transverse flexural and tensile tests, was still relatively modest.

Human protein aging: modification and crosslinking through dehydroalanine and dehydrobutyrine intermediates

Nonenzymatic post-translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC–tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty-four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through β-elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein–protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between βB2- & βB2-, βA4- & βA3-, γS- & βB1-, and βA4- & βA4-crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long-lived proteins and that DHA- and DHB-mediated protein crosslinking may be the source of the long-sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long-lived proteins of other tissues leading to protein aggregation diseases.

Impact of Acid and Alkaline Pretreatments on the Molecular Network of Wheat Gluten and on the Mechanical Properties of Compression-Molded Glassy Wheat Gluten Bioplastics

Wheat gluten can be converted into rigid biobased materials by high-temperature compression molding at low moisture contents. During molding, a cross-linked protein network is formed. This study investigated the effect of mixing gluten with acid/alkali in 70% ethanol at ambient temperature for 16 h followed by ethanol removal, freeze-drying, and compression molding at 130 and 150 °C on network formation and on types of cross-links formed. Alkaline pretreatment (0-100 mmol/L sodium hydroxide or 25 mmol/L potassium hydroxide) strongly affected gluten cross-linking, whereas acid pretreatment (0-25 mmol/L sulfuric acid or 25 mmol/L hydrochloric acid) had limited effect on the gluten network. Molded alkaline-treated gluten showed enhanced cross-linking but also degradation when treated with high alkali concentrations, whereas acid treatment reduced gluten cross-linking. β-Elimination of cystine and lanthionine formation occurred more pronouncedly at higher alkali concentrations. In contrast, formation of disulfide and nondisulfide cross-links during molding was hindered in acid-pretreated gluten. Bioplastic strength was higher for alkali than for acid-pretreated samples, whereas the flexural modulus was only slightly affected by either alkaline or acid pretreatment. Apparently, the ratio of disulfide to nondisulfide cross-links did not affect the mechanical properties of rigid gluten materials.

Proteomic Analysis of Temperature-Dependent Changes in Stored UHT Milk

Molecular changes in milk proteins during storage of UHT-treated milk have been investigated using two-dimensional electrophoresis (2-DE) coupled to MALDI-TOF mass spectrometry. UHT-treated samples were stored at three different temperatures, 4 °C, 28 °C, and 40 °C, for two months. Three main changes could be observed on 2-DE gels following storage. They were (1) the appearance of diffuse staining regions above the position of the monomeric caseins caused by nondisulfide cross-linking of α and β-caseins; (2) the appearance of additional acidic forms of proteins, predominantly of α(S1)-casein, caused by deamidation; and (3) the appearance of "stacked spots" caused by lactosylation of whey proteins. The extent of the changes increased with increased storage temperature. Mass spectrometric analysis of in-gel tryptic digests showed that the cross-linked proteins were dominated by α(S1)-casein, but a heterogeneous population of cross-linked forms with α(S2)-casein and β-casein was also observed. Tandem MS analysis was used to confirm deamidation of N(129) in α(S1)-casein. MS analysis of the stacked spots revealed lactosylation of 9/15 lysines in β-lactoglobulin and 8/12 lysines in α-lactalbumin. More extensive analysis will be required to confirm the nature of the cross-links and additional deamidation sites in α(S1)-casein as the highly phosphorylated nature of the caseins makes them challenging prospects for MS analysis.

Myeloperoxidase-dependent Inactivation of Surfactant Protein D in Vitro and in Vivo

Surfactant protein D (SP-D) plays diverse and important roles in innate immunity and pulmonary homeostasis. Neutrophils and myeloperoxidase (MPO) colocalized with SP-D in a murine bacterial pneumonia model of acute inflammation, suggesting that MPO-derived reactive species might alter the function of SP-D. Exposure of SP-D to the complete MPO-H(2)O(2)-halide system caused loss of SP-D-dependent aggregating activity. Hypochlorous acid (HOCl), the major oxidant generated by MPO, caused a similar loss of aggregating activity, which was accompanied by the generation of abnormal disulfide-cross-linked oligomers. A full-length SP-D mutant lacking N-terminal cysteine residues and truncation mutants lacking the N-terminal domains were resistant to the oxidant-induced alterations in disulfide bonding. Mass spectroscopy of HOCl-treated human SP-D demonstrated several modifications, but none involved key ligand binding residues. There was detectable oxidation of cysteine 15, but no HOCl-induced cysteine modifications were observed in the C-terminal lectin domain. Together, the findings localize abnormal disulfide cross-links to the N-terminal domain. MPO-deficient mice showed decreased cross-linking of SP-D and increased SP-D-dependent aggregating activity in the pneumonia model. Thus, MPO-derived oxidants can lead to modifications of SP-D structure with associated alterations in its characteristic aggregating activity.

Modification of surfactant protein D by reactive oxygen‐nitrogen intermediates is accompanied by loss of aggregating activity, in vitro and in vivo

Surfactant protein D (SP-D) is an important effector of innate immunity. We have previously shown that SP-D accumulates at sites of acute bacterial infection and neutrophil infiltration, a setting associated with the release of reactive species such as peroxynitrite. Incubation of native SP-D or trimeric SP-D lectin domains (NCRDs) with peroxynitrite resulted in nitration and nondisulfide cross-linking. Modifications were blocked by peroxynitrite scavengers or pH inactivation of peroxynitrite, and mass spectroscopy confirmed nitration of conserved tyrosine residues within the C-terminal neck and lectin domains. Mutant NCRDs lacking one or more of the tyrosines allowed us to demonstrate preferential nitration of Tyr314 and the formation of Tyr228-dependent cross-links. Although there was no effect of peroxynitrite or tyrosine mutations on lectin activity, incubation of SP-D dodecamers or murine lavage with peroxynitrite decreased the SP-D-dependent aggregation of lipopolysaccharide-coated beads, supporting our hypothesis that defective aggregation results from abnormal cross-linking. We also observed nitration, cross-linking of SP-D, and a significant decrease in SP-D-dependent aggregating activity in the lavage of mice acutely exposed to nitrogen dioxide. Thus, modification of SP-D by reactive oxygen-nitrogen species could contribute to alterations in the structure and function of SP-D at sites of inflammation in vivo.

Palisade is required in the Drosophila ovary for assembly and function of the protective vitelline membrane

The innermost layer of the Drosophila eggshell, the vitelline membrane, provides structural support and positional information to the embryo. It is assembled in an incompletely understood manner from four major proteins to form a homogeneous, transparent extracellular matrix. Here we show that RNAi knockdown or genetic deletion of a minor constituent of this matrix, Palisade, results in structural disruptions during the initial synthesis of the vitelline membrane by somatic follicle cells surrounding the oocyte, including wide size variation among the precursor vitelline bodies and disorganization of follicle cell microvilli. Loss of Palisade or the microvillar protein Cad99C results in abnormal uptake into the oocyte of sV17, a major vitelline membrane protein, and defects in non-disulfide cross-linking of sV17 and sV23, while loss of Palisade has additional effects on processing and disulfide cross-linking of these proteins. Embryos surrounded by the abnormal vitelline membranes synthesized when Palisade is reduced are fertilized but undergo developmental arrest, usually during the first 13 nuclear divisions, with a nuclear phenotype of chromatin margination similar to that described for wild-type embryos subjected to anoxia. Our results demonstrate that Palisade is involved in coordinating assembly of the vitelline membrane and is required for functional properties of the eggshell.

Glucose oxidase effect on dough rheology and bread quality: A study from macroscopic to molecular level

Enzymes are used in baking to improve dough handling properties and the quality of baked products. Glucose oxidase (GO) is an enzyme with oxidizing effect due to the hydrogen peroxide released from its catalytic reaction. In this study, the macroscopic effect of increasing glucose oxidase concentrations on wheat dough rheology, fresh bread characteristics and its shelf life during storage was determined. A reinforcement or strengthening of wheat dough and an improvement of bread quality can be obtained with the addition of GO, although inverse effects were obtained when excessive enzyme levels were added. The analysis of the gluten proteins at molecular level by high performance capillary electrophoresis and at supramolecular level by cryo-scanning electron microscopy revealed that the GO treatment modified gluten proteins (gliadins and glutenins) through the formation of disulfide and non-disulfide crosslinks. The high molecular weight glutenin subunits showed to be the most susceptible glutenin fraction to the oxidation action of GO. Excessive addition of GO produced an excessive crosslinking in the gluten network, responsible of the negative effect on the breadmaking properties.

Effect of cross-linking on the chaperone-like function of alpha crystallin

Alpha crystallin can function as a molecular chaperone in suppressing the heat-induced aggregation of other crystallins and proteins. During cataractogenesis, α-crystallin becomes a water-insoluble, high-molecular-weight, cross-linked aggregate. To determine whether the chaperone activity of alpha crystallin is lost during this age-related modification, extracts were prepared by sonication of water-insoluble proteins isolated from aged bovine lenses and human cataract lenses. All the preparations were tested for chaperone-like activity using βL-crystallin as the target protein and the percentage of α-crystallin in water-insoluble sonicated supernatant (WISS) was determined by slot blot immunoassay. The WISS from bovine as well as human lenses were still effective in protecting βL-crystallin aggregation at 56°C. The bovine cortical WISS with 50% immunoreactive α-crystallin showed 62% of the chaperone-like activity displayed by native α-crystallin. The WISS from bovine lens nucleus and human lenses with 17% and 5% immunoreactive α-crystallin showed 19% and 4% chaperone-like activity compared to native α-crystallin. Prior treatment of the WISS of both bovine and human lenses with dithiothreitol resulted in nearly 50% increase in chaperone-like activity suggesting possible loss of chaperone-like activity due to disulfide cross-links. To see if the chaperone-like activity of α-crystallin can be altered by non-disulfide cross-linking, native α-crystallin isolated from bovine lenses was cross-linked with dimethylsuberimidate (DMS) and dimethyl 3,3′-dithiobispropionimidate (DTBP) and tested for chaperone-like activity. The DMS cross-linked α-crystallin was effective in inhibiting the aggregation of βL-crystallins at 56°C, but required a two- to five-fold higher concentration than the native α-crystallin. α-Crystallin with higher degree of cross-linking showed lower chaperone-like activity. α-Crystallin cross-linked with DTBP, a cleavable cross-linking agent, also showed a 80% loss in chaperone-like activity. However, when the DTBP cross-linked α-crystallin was treated with dithiothreitol to cleave the cross-links there was a 50% recovery in the chaperone-like activity. These data suggest that the age-related cross-linking, which restricts the molecular flexibility of α-crystallin decreases its chaperone-like function.

Reductive Cleavage of the Disulfide Bonds of the Collagen IV Noncollagenous Domain in Aqueous Sodium Dodecyl Sulfate: Absence of Intermolecular Nondisulfide Cross-Links

The subunits of the collagen IV hexameric, noncollagenous NC1 domain obtained from bovine aorta, glomerular basement membrane, alveolar basement membrane and placental basement membrane are predominantly dimers. A large fraction of the dimers had been thought to be linked by nondisulfide bonds because they were resistant to cleavage by 5% (v/v) 2-mercaptoethanol, 2% (w/v) SDS, at 100°C. However, if an unusually high concentration of 2-mercaptoethanol, e.g., 40% (v/v), is used, complete conversion of dimers into monomers is achieved, indicating the lack of intersubunit nondisulfide cross-links. Electrophoresis patterns indicate that some of the intermolecular disulfide bonds of the dimers are more resistant to reduction in aqueous SDS than are some of the intramolecular disulfide bonds.

Isolation of insoluble secretory product from bovine thyroid: extracellular storage of thyroglobulin in covalently cross-linked form.

Extracellular storage of thyroglobulin (TG) is an important prerequisite for maintaining constant levels of thyroid hormones in vertebrates. Storage of large amounts is made possible by compactation of TG in the follicle lumen with concentrations of at least 100-400 mg/ml. We recently observed that the luminal content from bovine thyroids can be isolated in an intact state and be separated from soluble TG. For this purpose, bovine thyroid tissue was homogenized and subjected to various steps of purification. This procedure resulted in a pellet of single globules measuring 20-120 microns in diameter. Scanning electron microscopy revealed a unique cobblestone-like surface pattern of isolated globules, showing in detail the impressions of the apical plasma membranes of thyrocytes which had formerly surrounded the luminal content before tissue homogenization. Isolated thyroid globules were rapidly digested by trypsin but extremely resistant to various protein solubilization procedures. Homogenization of isolated globules resulted in the release of approximately 3% of total protein, showing that only a minor proportion of TG was loosely incorporated in thyroid globules whereas approximately 22% appeared to be interconnected with the globule matrix by disulfide bridges. Analysis by SDS-gel electrophoresis and immunoblotting confirmed that the protein released by this procedure consisted of TG. The vast majority (approximately 75%) of the globule matrix protein was found to be covalently cross-linked by non-disulfide bonds. TG in isolated globules was highly iodinated (approximately 55 iodine atoms per 12-S TG subunit) suggesting that the covalent nondisulfide cross-linking occurs in part during the iodination of TG and that this process involves the formation of intermolecular dityrosine bridges. Mechanisms must exist which solubilize or disperse the insoluble luminal content prior to endocytosis of TG.

Alveolar Basement Membrane: Molecular Properties of the Noncollagenous Domain (Hexamer) of Collagen IV and its Reactivity with Goodpasture Autoantibodies

The noncollagenous domain hexamer of collagen IV from bovine alveolar basement membrane was excised with bacterial collagenase, purified under nondenaturing conditions, and characterized. The hexamer is comprised of four distinct subunits [alpha 1(IV)NC1, alpha 2(IV)NC1, alpha 3(IV)NC1, and alpha 4(IV)NC1]. Each subunit exists in both monomeric and dimeric (disulfide-crosslinked) form, and both monomers and dimers have charge isoforms. Certain dimers also contain nonreducible crosslinks. The alpha 3(IV)NC1 subunit, in both the monomeric and dimeric form, reacts with Goodpasture (GP) antibodies. The GP epitope is sequestered within the hexamer and becomes reactive with antibody upon exposure with protein denaturants. These results reveal that the alveolar basement membrane hexamer is identical to the hexamer from glomerular basement membrane with respect to subunit composition, identity of subunits reacting with GP antibodies, and sequestration of the GP epitope but differs greatly in the relative amount of the GP-reactive subunit and the degree of disulfide and nondisulfide crosslinking of subunits. This study leads to the conclusion that pulmonary hemorrhage associated with GP syndrome is mediated by the same autoantibody that mediates the glomerulonephritis, namely anti-collagen [alpha 3(IV)] antibody.

Formation of pentosidine during nonenzymatic browning of proteins by glucose. Identification of glucose and other carbohydrates as possible precursors of pentosidine in vivo

A fluorescent compound has been detected in proteins browned during Maillard reactions with glucose in vitro and shown to be identical to pentosidine, a pentose-derived fluorescent cross-link formed between arginine and lysine residues in collagen (Sell, D. R., and Monnier, V. M. (1989) J. Biol. Chem. 264, 21597-21602). Pentosidine was the major fluorophore formed during nonenzymatic browning of ribonuclease and lysozyme by glucose, but accounted for less than 1% of non-disulfide cross-links in protein dimers formed during the reaction. Pentosidine was formed in greatest yields in reactions of pentoses with lysine and arginine in model systems but was also formed from glucose, fructose, ascorbate, Amadori compounds, 3-deoxyglucosone, and other sugars. Pentosidine was not formed from peroxidized polyunsaturated fatty acids or malondialdehyde. Its formation from carbohydrates was inhibited under nitrogen or anaerobic conditions and by aminoguanidine, an inhibitor of advanced glycation and browning reactions. Pentosidine was detected in human lens proteins, where its concentration increased gradually with age, but it did not exceed trace concentrations (less than or equal to 5 mumol/mol lysine), even in the 80-year-old lens. Although its precise carbohydrate source in vivo is uncertain and it is present in only trace concentrations in tissue proteins, pentosidine appears to be a useful biomarker for assessing cumulative damage to proteins by nonenzymatic browning reactions with carbohydrates.

Role of fructose in glycation and cross-linking of proteins.

Incubation of carbohydrate-free human serum albumin (HSA) with fructose in an aqueous buffer at pH 7.4 resulted in glycation of epsilon-amino groups of lysyl residues. A recently developed procedure, involving analysis of hexitol amino acids by high-performance liquid chromatography of phenylthiocarbamyl derivatives, was used to show that 85% of the bound hexose was attached to protein via carbon 2 (C-2). The remainder was attached to protein via carbon 1 (C-1). When incubations were conducted with glucose under identical conditions, all the hexose was attached via C-1. Examination of human ocular lens proteins showed that the majority of the covalently bound hexose was connected to epsilon-amino groups of lysyl residues via C-1; this was attributed mainly to nonenzymatic glucosylation in vivo, which has already been documented. A significant proportion (10-20%) of the bound hexose was connected via C-2. In view of the HSA-hexose incubation results (above), this indicated that the lens proteins had reacted with endogenous fructose; i.e., they had undergone nonenzymatic fructosylation in vivo. The model protein bovine pancreatic ribonuclease A reacted with fructose and glucose at similar rates under physiological conditions. However, covalent, non-disulfide cross-linking, which could be inhibited by D-penicillamine, was induced 10 times more rapidly by fructose than by glucose. It is postulated that some of the protein cross-linking that occurs in vivo is fructose-induced. The possible significance of these processes in diabetic subjects is discussed.