Hydrolytic Fragments Research Articles

Ex vivo degradation of β-Casomorphin-7 by human plasma peptidases: Potential implications for peptide systemic effects

Susceptibility to proteolytic activity is a critical limitation for food-derived peptides possibly influencing human physiological processes. This study explores the ex vivo stability and degradation kinetics of the milk-derived opioid peptide β-casomorphin-7 (BCM7) in human blood. Blood specimens collected from three healthy volunteers were individually spiked with synthetic BCM7 and sampled at seven time points over 2 h. Liquid chromatography-electrospray-high resolution tandem mass spectrometry was used to monitor the stability of BCM7 and the formation of its hydrolytic fragments. Human plasma peptidases rapidly hydrolyzed BCM7 generating inactive peptides with similar sharp degradation kinetics across the blood of different individuals. The estimated plasma half-life (t1/2) value of BCM7 ranged from 35 to 40 min. The peptide degradation pattern pointed to prolyl oligopeptidase, prolidase, and dipeptidyl peptidase as the primary enzyme candidates responsible for BCM7 hydrolysis. Overall, the findings of this study suggest that BCM7 cannot exert systemic effects in humans.

Base excision repair of the N-(2-deoxy-d-erythro-pentofuranosyl)-urea lesion by the hNEIL1 glycosylase.

The N-(2-deoxy-d-erythro-pentofuranosyl)-urea DNA lesion forms following hydrolytic fragmentation of cis-5R,6S- and trans-5R,6R-dihydroxy-5,6-dihydrothymidine (thymine glycol, Tg) or from oxidation of 7,8-dihydro-8-oxo-deoxyguanosine (8-oxodG) and subsequent hydrolysis. It interconverts between α and β deoxyribose anomers. Synthetic oligodeoxynucleotides containing this adduct are efficiently incised by unedited (K242) and edited (R242) forms of the hNEIL1 glycosylase. The structure of a complex between the active site unedited mutant CΔ100 P2G hNEIL1 (K242) glycosylase and double-stranded (ds) DNA containing a urea lesion reveals a pre-cleavage intermediate, in which the Gly2 N-terminal amine forms a conjugate with the deoxyribose C1' of the lesion, with the urea moiety remaining intact. This structure supports a proposed catalytic mechanism in which Glu3-mediated protonation of O4' facilitates attack at deoxyribose C1'. The deoxyribose is in the ring-opened configuration with the O4' oxygen protonated. The electron density of Lys242 suggests the 'residue 242-in conformation' associated with catalysis. This complex likely arises because the proton transfer steps involving Glu6 and Lys242 are hindered due to Glu6-mediated H-bonding with the Gly2 and the urea lesion. Consistent with crystallographic data, biochemical analyses show that the CΔ100 P2G hNEIL1 (K242) glycosylase exhibits a residual activity against urea-containing dsDNA.

IDE Degrades Nociceptin/Orphanin FQ through an Insulin Regulated Mechanism.

Insulin-degrading enzyme (IDE) was applied to catalyze hydrolysis of Nociceptin/Orphanin 1-16 (OFQ/N) to show the involvement of the enzyme in degradation of neuropeptides engaged in pain transmission. Moreover, IDE degradative action towards insulin (Ins) was inhibited by the OFQ/N fragments, suggesting a possible regulatory mechanism in the central nervous system. It has been found that OFQ/N and Ins affect each other degradation by IDE, although in a different manner. Indeed, while the digestion of OFQ/N is significantly affected by the presence of Ins, the kinetic profile of the Ins hydrolysis is not affected by the presence of OFQ/N. However, the main hydrolytic fragments of OFQ/N produced by IDE exert inhibitory activity towards the IDE-mediated Ins degradation. Here, we present the results indicating that, besides Ins, IDE cleaves neuropeptides and their released fragments act as inhibitors of IDE activity toward Ins. Having in mind that IDE is present in the brain, which also contains Ins receptors, it cannot be excluded that this enzyme indirectly participates in neural communication of pain signals and that neuropeptides involved in pain transmission may contribute to the regulation of IDE activity. Finally, preliminary results on the metabolism of OFQ/N, carried out in the rat spinal cord homogenate in the presence of various inhibitors specific for different classes of proteases, show that OFQ/N proteolysis in rat spinal cord could be due, besides IDE, also to a cysteine protease not yet identified.

Kinetics and Characterization of Non-enzymatic Fragmentation of Monoclonal Antibody Therapeutics.

To understand non-enzymatic hydrolytic fragmentation of a monoclonal antibody therapeutic under temperature stressed conditions and investigating possible mechanism for the same. The mAb therapeutic was incubated at 50°C in phosphate buffer at pH6.5 and fragmentation was monitored at different ionic strengths under stressed conditions. The incubated mAb was sampled at regular time intervals by analytical Size Exclusion Chromatography (SEC). It was observed that 57% of the mAb product fragmented over 4days into two fragment species - Fc-Fab and Fab with molecular weights of 97 KDa and 47 KDa, respectively, as measured by mass spectrometry (MS) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The fragmentation rate was slow initially and then accelerated with time. No change in % aggregate level was observed in this duration, implying that degradation was primarily via fragmentation at high temperature. Kinetics of hydrolytic fragmentation was hypothesized and SEC data was fitted to estimate the kinetic rate constants. While degradation of the monomer into fragment species was non-Arrhenius with a negative activation energy, further degradation of Fab-Fc fragments into Fab or Fc fragments followed Arrhenius Law with an activation energy of 2.1 and 15.38kcal/mol, respectively. High temperature (50°C) causes mAb to cleave at the hinge region to form Fab-Fc and Fab/Fc, as confirmed by dynamic light scattering, SDS-PAGE, SEC, and MS. A kinetic model for hydrolytic fragmentation has been proposed. The results are expected to assist end users in formulation development as well as in monitoring stability of biotherapeutic products.

Fragmentation of Injectable Bioadhesive Hydrogels Affords Chemotherapeutic Macromolecules.

Implantation of drug delivery depots into or proximal to targeted tissue is an effective method to deliver anticancer drugs in a sustained localized manner. Herein, syringe-injectable polydextran aldehyde (PDA)-based bioadhesive gels are prepared that can locally deliver cytotoxins upon their hydrolytic fragmentation. Adhesive gels are formed by mixing doxorubicin (DOX)-functionalized PDA (DOX-PDA) and bovine serum albumin (BSA) using a dual-barrel syringe. Upon mixing and delivery, the DOX-PDA reacts with the cross-linker BSA as well as the extracellular matrix via imine bond formation to define the cohesive and adhesive properties of the gel, respectively. Resulting gels are mechanically rigid (∼10 kPa) and adherent (adhesive stress ∼ 4 kPa). Once formed, the DOX-PDA-BSA gels undergo slow hydrolytic degradation (>2 months) locally releasing free DOX and DOX-PDA as expected. Surprisingly, we found that macromolecules composed of DOX, PDA, and BSA are also released from the bulk material. These DOX-PDA-BSA macromolecules, along with free DOX and DOX-PDA conjugate, are internalized by A549 lung carcinoma cells, resulting in potent cell death.

Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides

Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of l-tyrosine ethyl ester in aqueous media at 30°C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75mM, respectively. In the reaction initiated with 100mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15mM, the reaction proceeded poorly; however, from 30mM to 120mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, l-tyrosine ethyl ester, even at 5mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes l-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate.

Release of wheat gluten exorphins A5 and C5 during in vitro gastrointestinal digestion of bread and pasta and their absorption through an in vitro model of intestinal epithelium

The release of wheat gluten exorphins (GEs) A5, A4, B5, B4, C5, Gd-7 and dGd-7 during in vitro gastrointestinal digestion (GID) of bread and pasta samples has been investigated by UPLC/ESI–HRMS. The GEs that survived in vitro GID were studied for transport studies across monolayers of 70% Caco-2/30% HT-29 co-culture grown on semi-permeable membranes, used as a model of the human intestinal epithelium. Among the GEs, only the peptides A5 and C5 were detected at the end of in vitro GID of bread and pasta samples. Levels of GEs A5 and C5 were in the range 0.747–2.192mgkg−1 and 3.201–6.689mgkg−1, respectively. The absorption behavior of A5 and C5 was studied in detail using synthetic peptides. The two peptides were susceptible to the action of Caco-2/HT-29 co-culture peptidases, and 3% and 1% of A5 and C5, respectively, added to the apical chamber were transported to the basolateral chamber after 120min of incubation, without being hydrolysed. Several hydrolytic fragments of both GEs crossed the co-culture layer and were found in the basolateral chamber. For the first time, these results demonstrate that GEs are released during in vitro GID of real wheat-derived foods and highlight the possibility of transport of the GEs A5 and C5 across the human intestinal mucosa.

Polyphenolic C-glucosidic ellagitannins present in oak-aged wine inhibit HIV-1 nucleocapsid protein

HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone implicated in several steps of the virus replication cycle and an attractive new target for drug development. In reverse transcription, NC destabilizes nucleic acid secondary structures and catalyzes the annealing of HIV-1 TAR RNA to its DNA copy (cTAR) to form the heteroduplex TAR/cTAR. A screening program led to the identification of the plant polyphenols acutissimins A and B as potent inhibitors of NC in different assays. These two flavano-ellagitannins, which are found in wine aged in oak barrels, exhibited different mechanisms of protein inhibition and higher potency relatively to their epimers, epiacutissimins A and B, and to simpler structures notably representing hydrolytic fragments and metabolites therefrom.

Preferential and selective degradation and removal of amelogenin adsorbed on hydroxyapatites by MMP20 and KLK4 in vitro.

The hardest tooth enamel tissue develops from a soft layer of protein-rich matrix, predominated by amelogenin that is secreted by epithelial ameloblasts in the secretory stage of tooth enamel development. During enamel formation, a well-controlled progressive removal of matrix proteins by resident proteases, Matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), will provide space for the apatite crystals to grow. To better understand the role of amelogenin degradation in enamel biomineralization, the present study was conducted to investigate how the adsorption of amelogenin to hydroxyapatite (HAP) crystals affects its degradation by enamel proteinases, MMP20 and KLK4. Equal quantities of amelogenins confirmed by protein assays before digestions, either adsorbed to HAP or in solution, were incubated with MMP20 or KLK4. The digested samples collected at different time points were analyzed by spectrophotometry, SDS-PAGE, high performance liquid chromatography (HPLC), and LC-MALDI MS/MS. We found that majority of amelogenin adsorbed on HAP was released into the surrounding solution by enzymatic processing (88% for MMP20 and 98% for KLK4). The results show that as compared with amelogenin in solution, the HAP-bound amelogenin was hydrolyzed by both MMP20 and KLK4 at significantly higher rates. Using LC-MALDI MS/MS, more accessible cleavage sites and hydrolytic fragments from MMP20/KLK4 digestion were identified for the amelogenin adsorbed on HAP crystals as compared to the amelogenin in solution. These results suggest that the adsorption of amelogenin to HAP results in their preferential and selective degradation and removal from HAP by MMP20 and KLK4 in vitro. Based on these findings, a new degradation model related to enamel crystal growth is proposed.

UV photodegradation of murine growth hormone: Chemical analysis and immunogenicity consequences

During manufacturing, therapeutic proteins may be exposed to ultraviolet (UV) radiation. Such exposure is of concern because UV radiation may cause photooxidative damage to proteins, which in turn could lead to physical changes such as aggregation and enhanced immunogenicity. We exposed murine growth hormone (mGH) to controlled doses of UV radiation, and examined the resulting chemical, physical and immunogenic changes in the protein. mGH chemical structure was analyzed by mass spectrometry after UV irradiation. Photooxidation products detected by mass spectrometry included methionine sulfoxide formed at Met[127] and Met[149] residues, and, tentatively assigned by MS/MS analysis, ether cross-links between original Ser[78] and Cys[188], and Cys[206] and Ser[213], and a thioether cross-link between Cys[17] and Cys[78] residues, transformation of Cys[189] into Ala, and various hydrolytic fragments. Physical damage to UV-irradiated mGH was monitored by infrared spectrometry, chromatographic analyses, and particle counting by micro-flow imaging. UV radiation caused mGH to aggregate, forming insoluble microparticles containing mGH with non-native secondary structure. When administered subcutaneously to Balb/c or Nude Balb/c mice, UV-irradiated mGH provoked antibodies that cross-reacted with unmodified mGH in a fashion consistent with a T-cell dependent immune response. In wildtype Balb/c mice, titers for anti-mGH IgG1 antibodies increased with increasing UV radiation doses.

The chemistry of anti-pernicious anaemia factors; the ninhydrin-reacting hydrolytic fragment of vitamin B12.

Summary and Conclusions Our previous conclusion that acid hydrolysis of vitamin B12 leads to the formation of a “ninhydrin-reacting” substance is confirmed. The behaviour of this “ninhydrin-reacting” substance and of 2-aminopropanol on paper chromatograms has been examined. The results appear to show that these two substances are identical. Evidence has been obtained for the presence in acid hydrolysates of vitamin B12 of material showing selective absorption with bands and inflections at 2850, 2768, 2690, 2585 and 2500 å (referred to as “the 285 component“). This “285 component” is resolved into at least two structurally related substances by chromatography employing n-butyl alcohol-acetic acid as the irrigation solvent. Formation of ammonia occurs during acid or alkaline hydrolysis of vitamin B12. The authors thank Mr. R. E. Rodway for technical assistance in the isolation of crystalline vitamin B12 from a concentrate kindly supplied to the Research Department by Mr. A. W. Davidson. The ultra-violet absorption data were kindly determined by Dr. E. R. Holiday (M.R.C. Spectrographic Unit The London Hospital, E.1***), who also carried out the examinations with the Corning filter. The authors thank the Directors of The British Drug Houses, Ltd., for permission to publish these results.

The “Ninnydrin-Reacting” Hydrolytic Fragment of Vitamin B12

The “Ninnydrin-Reacting” Hydrolytic Fragment of Vitamin B12

Determination and Confirmation of Tulathromycin Residues in Bovine Liver and Porcine Kidney via Their Common Hydrolytic Fragment Using High-Performance Liquid Chromatography/Tandem Mass Spectrometry

An accurate, reliable, and reproducible analytical method using HPLC/MS/MS for the determination of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment (CP-60,300) was developed and validated. Briefly, the method involved an initial acid treatment of intact tissues, which yielded the common fragment (CP-60,300). A portion of the acid hydrolyzate was purified by SPE using a strong cation exchange cartridge. Evaporation of the purified extract was followed by reconstitution in aqueous buffer and analysis by HPLC/MS/MS under isocratic conditions. The developed method provided acceptable sensitivity for determinative surveillance of tulathromycin in porcine kidney and bovine liver with an LOQ of 7.50 and 2.75 microg/g, respectively. The overall recovery and precision of 45 determinations of each tissue were 97.8% (5.3%) for porcine kidney and 96.9% (7.9%) for bovine liver. Accuracy, precision, linearity, specificity, and ruggedness were demonstrated. An HPLC/MS/MS method was also developed for use in these tissues as a confirmatory assay following modifications to the MS detection parameters. The confirmatory method demonstrated acceptable sensitivity for confirmatory evaluation of tulathromycin in porcine kidney and bovine liver at tolerances of 15 and 5.5 microg/g, respectively.

Preservation of cell structures in a medieval infant brain: A paleohistological, paleogenetic, radiological and physico-chemical study

Cerebral tissues from archaeological human remains are extremely rare findings. Hereby, we report a multidisciplinary study of a unique case of a left cerebral hemisphere from a 13th century AD child, found in north-western France. The cerebral tissue–reduced by ca. 80% of its original weight–had been fixed in formalin since its discovery. However, it fully retained its gross anatomical characteristics such as sulci, and gyri; the frontal, temporal and occipital lobe as well as grey and white matter could be readily recognised. Neuronal remains near the hippocampus area and Nissl bodies from the motor cortex area were observed (Nissl, Klüver–Barrera staining). Also, computed tomography (CT) and magnetic resonance imaging (T1, proton density, ultra short echo time sequences) were feasible. They produced high quality morpho-diagnostic images. Both histological and radiological examinations could not confirm the pathologist's previously suggested diagnosis of cerebral haemorrhage as the cause of death. Reproducible cloned mtDNA sequences were recovered from the skeleton but not from the brain itself. This was most likely due to the combined effect of formaldehyde driven DNA–DNA and/or DNA–protein cross-linking, plus hydrolytic fragmentation of the DNA. The chemical profile of the brain tissue, from gas-chromatography/mass-spectroscopy analysis, suggested adipocerous formation as the main aetiology of the mummification process. The hereby presented child brain is a unique paleo-case of well-preserved neuronal cellular tissue, which is a conditio sine qua non for any subsequent study addressing wider perspectives in neuroscience research, such as the evolution of brain morphology and pathology.

Bromination/Hydrolytic Fragmentation Reactions of α,β-Unsaturated N-Boc Lactams for the Synthesis of ω-Amino-trisubstituted (E)-Vinyl Bromides

Scheme 2. Bromination/hydrolytic fragmentation reactions of N-Boc lactams 12a 12d. Trisubstituted (E)-halo alkenes are important synthetic building blocks frequently employed in the synthesis of many natural products including scyphostatin, octalactin, phomactins, borrelidin, apoptolidin, FK901464, phorboxazole A, fostriecin, taxifolial A, kendomycin, tedanolide and deoxytedanolide. We have previously developed and reported the bromination/hydrolytic fragmentation reactions of lactones 1 to trisubstituted (E)-vinyl bromides 3. As a scalable alternative to the conventional methods, this new strategy was successfuly applied to the gram-scale synthesis of enantiomerically pure trisubstituted (E)-vinyl bromide (-)-5 used as the C8-C11 fragment in the total synthesis of tedanolide (Scheme 1). In the reaction sequence, the putative intermediate 2c undergoes a C-C bond rotation for anti-coplanar alignment of the departing carboxylate and bromide groups before the elimination reaction, thus generating (E)-vinyl bromides in a highly stereoselective manner. Noticing the potential utility of ω-amino-trisubstituted (E)vinyl bromides (vide infra) for the synthesis of indolizidine and quinolizidine type alkaloid natural products, we decided to apply the above bromination/hydrolytic fragmentation protocol to α,β-unsaturated lactam system. To this end, a short array of α,β-unsaturated N-Boc lactams 12a 12d were prepared from the corresponding ω-amino aldehydes by following the literature procedures. Subsequent bromination reactions gave rise to trans-dibromides 13a 13d (75 ~ 85% yield), setting the stage for the ensuing hydrolytic fragmentation reactions. When treated with LiOH in aqueous DMF at rt, dibromides 13a 13d readily underwent the fragmentation reactions to provide the corresponding ω-amino-(E)-vinyl bromides 14a 14d, at a rate much faster than lactone dibromides 1 (Scheme 2). In summary, the bromination/hydrolytic fragmentation reactions on α,β-unsaturated N-Boc lactams furnished various previously unknown ω-amino-trisubstituted (E)-vinyl bromides, in a stereoselective manner and good overall yields. Their utility towards the synthesis of indolizidine and quinolizidine type alkaloid natural products are currently under investigation.

Mechanical properties of mammalian and fish gelatins based on their weight average molecular weight and molecular weight distribution

Acid porcine skin gelatins (type A), lime bone gelatins (type B) and gelatin from different cold water fish species were compared on the basis of low deformation mechanical properties, Bloom value, weight average molecular weight, molecular weight distribution and isoelectric point. The dynamic storage modulus and Bloom value for all types of gelatin increased with increasing weight average molecular weight. Type A and type B gelatins with similar weight average molecular weight exhibited different dynamic storage modulus (G′) and different Bloom values. This is most probably due to a different molecular weight distribution as well as the presence of different hydrolytic fragments. The present study suggests that it may be possible to improve the mechanical properties by removing low molecular weight molecules from a gelatin sample. The Bloom values for gelatin from haddock, saithe and cod were determined to be 200, 150 and 100 g from a linear correlation between G′ and Bloom.

Hydrolytic fragmentation of seed gums under microwave irradiation

The seed gum solutions of Ipomoea purga, Ipomoea palmata, Ipomoea dasysperma, Cyanaposis tetragonolobus (Guar gum) and Crotolaria medicaginea were microwave (MW) irradiated and their degradation to oligo and monosaccharides was investigated. The gum solutions were fragmented into oligosaccharides/constituent monosaccharides depending upon the length of MW exposure in presence of catalytic amount of mineral acid or even when no acid was used. A mechanism for the microwave induced hydrolytic degradation of the seed gums has been proposed. The MW exposure time required for the partial and complete degradation of the gums was found dependent on the types of the linkages and degree of the branching present in the gums.

Chondroitin sulphate decreases collagen synthesis in normal and scleroderma fibroblasts through a Smad‐independent TGF‐β pathway – implication of C‐Krox and Sp1

Despite several investigations, the transcriptional mechanisms which regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. In this study, we determined the effects of both native ichtyan chondroïtin sulphate (CS) and its derived hydrolytic fragments (CSf) on human normal (NF) and scleroderma (SF) fibroblasts. Here, we demonstrate for the first time that CS and CSf exert an inhibitory effect on type I collagen protein synthesis and decrease the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in NF and SF. These glycosaminoglycan molecules repress COL1A1 gene transcription through a -112/-61 bp sequence upstream the start site of transcription and imply hc-Krox and Sp1 transcription factors. In addition, CS and CSf induced a down-regulation of TβRI expression. As a conclusion, our findings highlight a possible new role for CS and CSf as anti-fibrotic molecules and could help in elucidating the mechanisms of action by which CS and CSf exert their inhibitory effect on type I collagen synthesis.

Eisenia fetida Protease-III-1 Functions in Both Fibrinolysis and Fibrogenesis

The fibrinolytic function of earthworm protease-III-1 (Ef P-III-1) has been studied in recent years. Here, we found that Ef P-III-1 acted not only in fibrinogenolysis, but also in fibrogenesis. We have used Ef P-III-1 to hydrolyze fibrinogen, and to activate plasminogen and prothrombin. Based on the N-terminal sequences of the hydrolytic fragments, Ef P-III-1 was showed to specifically recognize the carboxylic sites of arginine and lysine. Analyses by fibrinogenolysis mapping and amino acid sequencing revealed that the isozyme could cleave the alpha, beta, and gamma chains of fibrinogen, showing a high α-fibrinogenase, moderate β-fibrinogenase, and low γ-fibrinogenase activities. Interestingly, Ef P-III-1 activated plasminogen and released active plasmin, suggesting a tPA-like function. Furthermore, Ef P-III-1 showed a factor Xa-like function on prothrombin, producing alpha-thrombin. The function in both activating prothrombin and catalyzing fibrinogenolysis suggests that Ef P-III-1 may play a role in the balance between procoagulation and anticoagulation.

A Scalable Route to Trisubstituted (E)‐Vinyl Bromides.

AbstractFor Abstract see ChemInform Abstract in Full Text.