Molecular Recognition and Chemical Modification of Biopolymers — Two Main Components of Affinity Modification
Dmitri G. Knorre, Valentin V. Vlassov in Affinity Modification of Biopolymers, 1989
A special group of cross-linking reagents is represented by the so-called “contact site” or “zero length” cross-linking reagents68 which share the common property of being able to cross-link only very closely juxtaposed residues, giving rise to cross-links of a length so short as to imply that the cross-linked atoms must have been capable of coming into virtual contact with the native biopolymer structure. A typical representative of a contact site crosslinking reagent is formaldehyde which introduces one carbon atom bridge between the cross-linked amino groups and is referred to as a one-atom-bridge cross-linker. In this case, the distance between the covalently bridged atoms is similar to the minimal Van der Waals separation of the atoms before the modification. Formaldehyde is characterized by a very broad specificity allowing this reagent to be used for protein-protein and protein-nucleic acid cross-linking. It reacts readily with amino groups of protein side chains and heterocyclic bases of nucleic acids and cross-links them to amino groups or side chains of Glu, Asp, Cys, Tyr, Trp, His, or Arg. The main pathway of cross-linking includes hydroxymethylation of lysine residues followed by reactions with nucleophilic amino acid side chains.
The Chemical Cross-Linking of Peptide Chains
Roger L. Lundblad, Claudia M. Noyes in Chemical Reagents for Protein Modification, 1984
An interesting pair of reagents (Figure 3) which cross-link proteins by consecutive Michael reactions have been described.9 These investigators made the point that since the cross-linkage reaction is driven by consecutive Michael additions, eventually the most thermodynamically stable cross-link will be established, which can be subsequently stabilized by reduction of the nitro function with sodium dithionite. These investigators explored the reaction of pancreatic ribonuclease with 2-(p-nitrophenyl) allyl-4-nitro-3-carboxyphenyl sulfide (twofold molar excess with respect to ribonuclease in 0.1 M sodium phosphate, pH 10.5 at 37°C, 36 hr, cross-link stabilized by sodium dithionite fivefold molar excess). No reaction occurred at pH 8.0. Analysis of the reaction mixture showed 61% monomer, 21% dimer, 10% trimer, and a trace of tetramer. The monomer fraction was characterized; the predominant cross-links occurred between lysine-7 and lysine-37 and between lysine-31 and lysine-41.
The Chemical Cross-Linking Of Peptide Chains
Roger L. Lundblad in Chemical Reagents for Protein Modification, 2020
An interesting pair of reagents (Figure 2) which cross-link proteins by consecutive Michael reactions have been described.18a These investigators made the point that since the cross-linkage reaction is driven by consecutive Michael additions, eventually the most thermodynamically stable cross-link will be established, which can be subsequently stabilized by reduction of the nitro function with sodium dithionite. These investigators explored the reaction of pancreatic ribonuclease with 2-(p-nitrophenyl) allyl-4-nitro-3-carboxyphenyl sulfide (twofold molar excess with respect to ribonuclease in 0.1 M sodium phosphate, pH 10.5 at 37°C, 36 h, cross-link stabilized by sodium dithionite fivefold molar excess). No reaction occurred at pH 8.0. Analysis of the reaction mixture showed 61% monomer, 21% dimer, 10% trimer, and a trace of tetramer. The monomer fraction was characterized; the predominant cross-links occurred between lysine-7 and lysine-37 and between lysine-31 and lysine-41.
Thermo-responsive injectable naringin-loaded hydrogel polymerised sodium alginate/bioglass delivery for articular cartilage
Published in Drug Delivery, 2021
Xiang Li, Yang Lu, Yuxin Wang, Shengji Zhou, Liangping Li, Fengchao Zhao
Figure 1 shows a microscopic graphical representation of the Naringin–BG hydrogel gelling process. Three forms of crossed-link, like calcium-ion crossed-link, physical cross-link, and hydrogen crossed links, are primarily used in the gelation process. The interaction between the calcium ion and the slowly released BG calcium ion is triggered by sodium alginate binding. In addition, hydrogen links between alginate and agarose are established as the temperature changes (Liu et al., 2018). In Figure 2(A,B), the SEM picture showing the Naringin–BG hydrogel microstructure after lyophilization. Its key characteristic is a porous system representing cell growth, nutrient exchange and metabolites waste excretion with the outside world. Numerous bioactive glass fragments fitted on the hydrogel's pores. Figure 2(C) reveals that it took approximately 120 min to achieve equilibrium swelling of the Naringin–BG hydrogel samples in PBS solutions. The last swelling capacity for the Naringin–BG hydrogels lyophilized was 15. A first compound explosion of approximately 10% was shown in Figure 2(D). The hydrogel of Naringin–BG shows Figure 2(E) showed that three consecutive days of observation showed that Naringin stimulated chondrocyte proliferation with a concentration of 10 μM. In contrast, 5 μM Naringin did not have a clear impact, and 20 μM had a proliferation inhibition at 48 h. We use 30 times the efficient Naringin concentration in preparing Naringin–BG hydrogel to confirm the substance has adequate drug concentrations when added.
Chitosan-based nanotherapeutics for ovarian cancer treatment
Published in Journal of Drug Targeting, 2019
Leila Alizadeh, Amir Zarebkohan, Roya Salehi, Amir Ajjoolabady, Mohammad Rahmati-Yamchi
The most common method for preparation of CS NPs is a cross-linking technique. In this method, positively charged NH2 groups of chitosan cross-link with aldehyde group of cross-linker like glutaraldehyde [50]. Chitosan aqueous solution emulsified in an oil phase to prepare a water-in-oil (w/o) emulsion. Then, an appropriate surfactant was added to stabilise aqueous droplets. Thereafter, a suitable cross-linking agent such as glutaraldehyde was added to cross-link and harden droplets. Finally, microspheres should be filtered and washed repeatedly with n-hexane and alcohol and dry [4]. Chitosan/cross-linker ratio strongly affects the physicochemical properties of chitosan NPs including size, zeta potential, degradation rate, drug release profile and swelling. In addition, time of exposure to the cross-linker has effect on the percentage of entrapment efficacy [53].
Synthesis and characterisation of aqueous haemoglobin-based microcapsules coated by genipin-cross-linked albumin
Published in Journal of Microencapsulation, 2020
Kai Melvin Schakowski, Jürgen Linders, Katja Bettina Ferenz, Michael Kirsch
Genipin-induced polymerisation of BSA was observable by the change of colour of a shaken solution containing genipin and BSA. The solution of 5% BSA and 0.17% genipin equals a molar ratio of 1:10. At the beginning the solution presented a pale-yellow colour attributable to BSA. After 16 h of shaking at room temperature the colour had turned into a dark green, the combination of residue BSA monomer and the blue colour of oligomerised BSA described in literature (Touyama et al.1994, Butler et al.2003, Somers et al.2008, Yoo et al.2011). The change in colour was traceable by the appearance of a new signal in the spectrum of the BSA-genipin solution at 599 nm after the applied reaction period, as seen in Figure 3. The pure solution of BSA showed its highest absorbance in the area of blue and ultraviolet light, while the solution of pure genipin displayed hardly any absorbance in the spectrum of visible light. After merging equal volumes of the two solutions, the resulting spectrum presented the absorbance of a diluted solution of BSA. Cross-linking of BSA by genipin was additionally proven by comparing native PAGE traces of pure BSA and genipin-treated BSA to BSA cross-linked by glutaraldehyde, which is widely known to cross-link proteins (Xiong et al.2012, Shahgholian et al.2017). Cross-linking by genipin monitored by native PAGE resulted in a trace virtually identical to that of linking by glutaraldehyde (s. Figure 3).
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