Immobilization of lipase to chitosan beads using a natural cross-linker

Genipin, a reagent of plant origin was used for the immobilization of lipase by cross-linking to chitosan beads. The catalytic properties and operational and storage stabilities of the immobilized lipase were compared with the soluble lipase. Under optimum conditions, 198 microg protein was bound per g chitosan with a protein-coupling yield of 35%. The hydrolytic activity was 10.8 U/g chitosan and the relative specific activity was 108%. The immobilized lipase showed better thermal and pH stabilities compared to the soluble form. The immobilized enzyme exhibited mass transfer limitations as reflected by a higher apparent K(m) value and a lower energy of activation. The immobilized enzyme retained about 74% of its initial activity after five hydrolytic cycles.     

Preparation and characterization of a thermostable and biodegradable biopolymers using natural cross-linker

The present study describes preparation and characterization of a thermally stable and biodegradable biopolymer using collagen and a natural polymer, alginic acid (AA). Required concentration of alginic acid and collagen was optimized and the resulting biopolymer was characterized for, degree of cross-linking, mechanical strength, thermal stability, biocompatibility (toxicity) and biodegradability. Results reveal, the degree of cross-linking of alginic acid (at 1.5% concentration) with collagen was calculated as 75%, whereas it was 83% with standard cross-linking agent, glutaraldehyde (at 1.5% concentration). The AA cross-linked biopolymer was stable up to 245°C and Exhibits 5-6-fold increase in mechanical (tensile) strength compared to plain collagen (native) materials. However, glutaraldehyde cross-linked material exhibits comparatively less thermal stability and brittle in nature (low tensile strength). With regard to cell toxicity, no cytotoxicity was observed for AA cross-linked material when tested with mesenchymal cells and found degradable when treated with collagenase enzyme. The nature of bonding pattern and the reason for thermal stability of AA cross-linked collagen biopolymer was discussed in detail with the help of bioinformatics. A supplementary file on efficacy of AACC as a wound dressing material is demonstrated in detail with animal model studies.     

BiPS, a photocleavable, isotopically coded, fluorescent cross-linker for structural proteomics

Cross-linking combined with mass spectrometry is an emerging approach for studying protein structure and protein-protein interactions. However, unambiguous mass spectrometric identification of cross-linked peptides derived from proteolytically digested cross-linked proteins is still challenging. Here we describe the use of a novel cross-linker, bimane bisthiopropionic acid N-succinimidyl ester (BiPS), that overcomes many of the challenges associated with other cross-linking reagents. BiPS is distinguished from other cross-linkers by a unique combination of properties: it is photocleavable, fluorescent, homobifunctional, amine-reactive, and isotopically coded. As demonstrated with a model protein complex, RNase S, the fluorescent moiety of BiPS allows for sensitive and specific monitoring of the different cross-linking steps, including detection and isolation of cross-linked proteins by gel electrophoresis, determination of in-gel digestion completion, and fluorescence-based separation of cross-linked peptides by HPLC. The isotopic coding of BiPS results in characteristic ion signal "doublets" in mass spectra, thereby permitting ready detection of cross-linker-containing peptides. Under MALDI-MS conditions, partial photocleavage of the cross-linker occurs, releasing the cross-linked peptides. This allows differentiation between dead-end, intra-, and interpeptide cross-links based on losses of specific mass fragments. It also allows the use of the isotope doublets as mass spectrometric "signatures." A software program was developed that permits automatic cross-link identification and assignment of the cross-link type. Furthermore photocleavage of BiPS assists in cross-link identification by allowing separate tandem mass spectrometry sequencing of each peptide comprising the original cross-link. By combining the use of BiPS with MS, we have provided the first direct evidence for the docking site of a phosphorylated G-protein-coupled receptor C terminus on the multifunctional adaptor protein beta-arrestin, clearly demonstrating the broad potential and application of this novel cross-linker in structural and cellular biology.     

Modification of collagen in vitro with respect to formation of Nepsilon-carboxymethyllysine

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.     

Design of antiviral stapled peptides containing a biphenyl cross-linker

Here we report the design and synthesis of a panel of stapled peptides containing a distance-matching biphenyl cross-linker based upon a peptide capsid assembly inhibitor reported previously. Compared with the linear peptide, the biphenyl-stapled peptides exhibited significantly enhanced cell penetration and potent antiviral activity in the cell-based infection assays. Isothermal titration calorimetry and surface plasmon resonance experiments revealed that the most active stapled CAI peptide binds to the C-terminal domain of HIV capsid protein as well as envelop glycoprotein gp120 with low micromolar binding affinities, and as a result, inhibits both the HIV-1 virus entry and the virus assembly.     

Modes of genotoxicity of a macromolecular antibiotic, SN-07, a novel type of interstrand DNA cross-linker

The modes of genotoxicity of a novel macromolecular antitumor antibiotic (SN-07) were examined using both prokaryotic and eukaryotic cells in vitro. The antibiotic induced a frameshift-type reverse mutation in Ames Salmonella typhimurium TA98 at 1.6-400 ng/plate with and without S9 mix. SN-07 also induced chromosomal aberrations and a forward mutation (6-TGr) in Chinese hamster V79 cells after 1 h treatment at 12.5-100 ng/ml without metabolic activation. The alkaline elution technique revealed that SN-07 induced interstrand DNA cross-linking dose-dependently after treatment with 2.5-10 micrograms/ml for 1 h followed by elution at pH 12.1, but it did not induce the dose-dependent cross-linking after the same treatment followed by elution at pH 12.6. It was also found that SN-07 induced single-strand DNA breaks (pH 12.1) and alkali-labile (pH 12.6) sites after treatment with 0.1-10 micrograms/ml for 1 h followed by 24-h post-incubation.     

Phase transition in natural collagen]

The state of water protons and peptide chains in natural collagen was investigated by the method of nuclear magnetic resonance. The study of orientation and temperature dependence of collagen PMR spectra allows us to mage a conclusion about the considerable disorder of mutual orientation in the polypeptide chains at the temperature above -4 degrees C. Under these conditions it was observed that natural collagen was near to the liquid-crystal. The phasic transition at -4 degrees C was discovered, the latter was followed by an abrupt change of the character of water proton mobility and by the change of orientation of collagen fibres order. The thermal effect of transition was measured, the latter presents 18k/gr of the whole specimen.     

Modification of the side chain of micromolide, an anti-tuberculosis natural product

This paper describes a series of modifications of the side chain of micromolide, an anti-tuberculosis natural product. Most of the synthesized compounds showed significantly decreased activities, which suggests that the long aliphatic side chain of micromolide and its double bond are essential to its activity.     

Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling

Mammalian organs are typically comprised of several cell populations. Some (e.g. brain) are very heterogeneous, and this cellular complexity makes it difficult, if not impossible, to interpret expression profiles obtained with microarrays. Instruments, such as those manufactured by Leica or Arcturus, that permit laser capture microdissection of specific cells or cell groups from tissues were developed to solve this problem. To take full advantage of these instruments, however, one must be able to recognize cell populations of interest and, after they are harvested, to extract intact, unmodified RNA from them. Here we describe a novel, fast and simple method to fix and immunostain tissue sections that permits this to be done.     

Creation of cross-linked electrospun isotypic-elastin fibers controlled cell-differentiation with new cross-linker

Effective application of elastin materials for vascular grafts in tissue engineering requires these materials to retain the elastic and biological properties of native elastin. To clarify the influence of soluble elastin isotypes on vascular smooth muscle cells (VSMCs), soluble elastin was prepared from insoluble elastin by hydrolysis with oxalic acid. Its fractions were separated and classified into three isotypes. Elastin retaining 2.25 mol% of cross-linked structures exhibited significant differentiation of VSMCs, which adhered to the elastin with contraction phenotypes similar to that of native elastin, causing proliferation to cease. This trend was more strongly demonstrated in cotton-like elastin fibers with a new cross-linker. The results suggest that elastin isotypes could be applied as new effective biomaterials for suppressing intimal hyperplasia in vascular grafts.     

Conformational features of a natural break in the type IV collagen Gly-X-Y repeat

Fibrillar collagens have an absolute requirement for Gly as every 3rd residue, whereas breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domains of non-fibrillar collagens, such as type IV collagen in basement membranes. In this study, a model 30-mer peptide is designed to include the interruption GPOGAAVMGPOGPO found in the alpha5 chain of type IV collagen. The GAAVM peptide forms a stable triple helix, with Tm= 29 degrees C. When compared with a control peptide with Gly as every 3rd residue, the GAAVM peptide has a marked decrease in the 225 nm maximum of its CD spectrum and a 10 degrees C drop in stability. A 50% decrease in calorimetric enthalpy is observed, which may result from disruption of ordered water structure anchored by regularly placed backbone carbonyls. NMR studies on specific 15N-labeled residues within the GAAVM peptide indicate a normal triple helical structure for Gly-Pro-Hyp residues flanking the break. The sequence within the break is not disordered but shows altered hydrogen exchange rates and an abnormal Val chemical shift. It was previously reported that a peptide designed to model a similar kind of interruption in the peptide (Pro-Hyp-Gly)10, (GPOGPOPOGPO), is unable to form a stable triple helix, and replacement of GAA by GPO or VM by PO within the GAAVM break decreases the stability. Thus, rigid imino acids are unfavorable within a break, despite their favorable stabilization of the triple helix itself. These results suggest some non-random structure typical of this category of breaks in the Gly-X-Y repeat of the triple helix.     

Modification of native collagen reduces antigenicity but preserves cell compatibility

Porcine intestinal collagen (ICL), derived from processed small intestine, is used as a part of a remodelable bilaminate biosynthetic vascular prosthesis. The process for the production of ICL involves mechanical cleaning of non-crosslinked porcine intestine (NC-ICL), disinfection with peracetic acid (PA-ICL), and crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (PA/EDC-ICL). Two model systems were investigated to evaluate the effect of these agents on the humoral response to NC-ICL. First, the antibody titers of rabbits immunized with NC-ICL, PA-ICL, and PA/EDC-ICL were determined, and second, the humoral response of canines receiving collagenous vascular implants was examined. Collagenous and noncollagenous fractions were extracted from NC-ICL, PA-ICL, and PA/EDC-ICL and separated by SDS-PAGE. PA and EDC treatment decreased the number of extractable proteins as compared to NC-ICL. Immunoblot techniques demonstrated anti-NC-ICL antibodies recognized multiple immunoreactive proteins in NC-ICL, but not in PA-ICL or PA/EDC-ICL; and rabbits immunized with NC-ICL produced higher antibody titers to ICL proteins than rabbits immunized with either PA-ICL or PA/EDC-ICL. It was, therefore, apparent that NC-ICL was more antigenic than either PA-ICL or PA/EDC-ICL. The humoral immune response of canines to PA/EDC-ICL fabricated vascular grafts was determined. At 4 weeks, 8 weeks, and 12 weeks postimplant, serum antibodies to ICL proteins or type I collagen could not be detected. These data demonstrate a reduced humoral immune response to PA/EDC-ICL. (c) 1996 John Wiley & Sons, Inc.     

Synthesis and antioxidant activity of a procyanidin B3 analogue

Proanthocyanidin, an oligomer of catechin, is a natural antioxidant and a potent inhibitor of lectin-like oxidized LDL receptor-1, which is involved in the pathogenesis of arteriosclerosis. We synthesized proanthocyanidin analogue 1, in which the geometry of one catechin molecule in procyanidin B3, a dimer of (+)-catechin, is constrained to be planar. The antioxidant activities of the compounds were evaluated in terms of their capacities to scavenge galvinoxyl radicals, and results demonstrate that while procyanidin was 3.8 times more potent than (+)-catechin, the radical scavenging activity of proanthocyanidin analogue 1 was further increased to 1.9 times that of procyanidin B3. This newly designed proanthocyanidin analogue 1 may be a promising lead compound for the treatment of arteriosclerosis and related cerebrovascular diseases.     

A molecularly imprinted receptor for separation of testosterone and epitestosterone, based on a steroidal cross-linker

A series of molecularly imprinted polymers have been prepared and investigated as stationary phases in high performance liquid chromatography for the separation of testosterone and epitestosterone using non-polar mobile phases. The polymers were imprinted using 5α-dihydrotestosterone as template, and all retain testosterone more strongly than its 17α-OH epimer. The best polymer was prepared using trifluoromethylacrylic acid as functional monomer (interacting with the template via hydrogen bonds), divinylbenzene as ‘inert’ cross-linker, and chloroform as porogen. It also included a steroid-based cross-linker, which may interact with the template via van der Waals interactions to lend additional ‘shape selectivity’. A 250 × 4.6 mm column packed with this polymer gave baseline resolution of testosterone and epitestosterone (15 μg each) in under 20 min. Preparation of the steroid based cross-linker included the selective reduction of 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) to the 3α,17β-diol using K-selectride.     

Evidence for natural existence of pyridinoline crosslink in collagen

Pyridinoline is a fluorescent crosslinking amino acid isolated from collagen. Recently it was claimed that this material is an artefact produced from contaminating proteins during acid hydrolysis. However, in our hands, bovine tendon collagen could not be depleted of pyridinoline by the suggested treatments. A peptide which had the same fluorescence properties as those of pyridinoline could be isolated from enzymic digests of collagen. After acid hydrolysis, presence of pyridinoline in the peptide could be demonstrated on amino acid analysis. The composition of the peptide suggests that it originates from the specific regions of collagen molecule. These results clearly indicate the existence of pyridinoline in collagen $\text{in}\text{vivo}$.     

Synthesis of chemotactic peptide analogs with a photoaffinity cross-linker as new probes for analysis of receptor-ligand interaction

Formylpeptide receptors are well-characterized receptors which participate in host defense responses of neutrophils. We designed and synthesized chemotactic peptide analog with p-benzoylphenylalanine (Bpa) and biotin to probe structural and mechanistic aspects of peptide-receptor interaction. These peptides possess biological activities which were dependent upon spacer residue length of and Bpa position. The covalent photoaffinity label was detected by Streptavidine-blot, which was inhibited by the parent peptide.     

The glue protein of ribbed mussels (Geukensia demissa): a natural adhesive with some features of collagen

Summary The Atlantic ribbed musselGeukensia (Modiolus)demissa attaches itself to the roots of cord grass and other hard objects in tidal salt marshes by spinning adhesive byssal threads. The precursor of a protein apparently present in the adhesive plaques of the threads was isolated in quantity from the foot of the mussel. The protein has an apparent molecular weight of 130000, a pI of 8.1, and contains a high proportion of Gly, Glu/Gln, Lys and 3,4-dihydroxyphenyl-l-alanine (DOPA). Sequence of tryptic peptides suggests a pattern of repeated motifs, such as: Gly-DOPA-Lys, and X-Gly-DOPA-Y-Z-Gly-DOPA/Tyr-Lys, where X is Thr or Ala in octapeptides and Gln-Thr in nonapeptides. Y is variable, but more often than not hydrophobic; and Z is frequently Pro or 4-trans-hydroxyproline (Hyp). The presence of Pro-Gly and Hyp-Gly sequences of -hydroxylysine in the protein is reminiscent of typical collagens; however, the protein is not labile to clostridial collagenase, nor does collagen cross-react with antibodies raised against the mussel protein. Unlike typical collagens, Gly probably occurs only at every 4th or 5th residue in this unusual mussel protein.Abbreviations Anti-Gdfp anti-G. demissa foot protein - Dopa 3,4-dihydroxyphenylalanine - CTAB cetyltrimethylammonium bromide - HPLC high performance liquid chromatography - PAGE polyacrylamide gel electrophoresis Supported by grants from the National Science Foundation (DMB 8500301) and the Office of Naval Research (N00014-86K-0717)