The Covalent Binding of Genistein to the Non-prosthetic-heme-moiety of Bovine Lactoperoxidase Leads to Enzymatic Inactivation

Objective Genistein, a major soy isoflavone metabolite （SIF）, inactivates oxidation activity of bovine lactoperoxidase （LPO）. Modification of the heme moiety of LPO by nitrogen-containing compounds has been shown to inactivate LPO. In contrast, SIF mediated inactivation of LPO does not involve a heme modification and the mechanism of SIF inhibition is poorly understood. Methods After inactivation of LPO by genistein in the presence of H202, trypsin-digested LPO peptide fragments were collected and analyzed by MALDI-TOF-MS to characterize the chemical binding of genistein（s） to LPO. Results The heme moiety of LPO was not modified by genistein. A covalent binding study showed that 3H-genistein bound to LPO with a ratio of ~12 to 1. After HPLC analysis and peak collection, trypsin-digested peptide fragments were analyzed by MALDI-TOF-MS. The 3H-genistein co-eluted peptide fragments （RT=24 min） were putatively identified as 1991VGYLDEEGVLDQNR214 with two bound genistein molecules or a genistein dimer （2 259 Da）, 486TPDNIDIWlGGNAEPMVER504 with two bound genistein molecules or a genistein dimer （2 663 Da）, and 161ARWLPAEYEDGLALPFGWTQR182 with three bound genistein molecules or a genistein trimer （3 060 Da）. The fragment with a mass of 2 792 Da （RT=36 min） was identified as 132CDENSPYR139 with three genistein molecules or a genistein trimer. Conclusions The results suggest that LPO was inactivated by irreversible covalent binding of genistein or genistein polymers to particular peptide fragments constituting regions of the outward domain. No genistein interaction with the prosthetic heme moiety of LPO was observed.

Immobilization of Lipase by Covalent Binding on Crosslinked Allyl Dextran

Lipase was immobilized by covalent binding on crosslinked allyl dextranusing SESA as coupling agent. It is shown that this immobilization approach isan efficient one for lipase. The activity of the immobilized lipase can rerch to300 - 450 U/g (dry weight). It exhibits good temperature stability, can retain88% activity after being incubated at 70℃ for 2 h. Special effects will be expected from our immobilized lipase in its applications in organic media due tothe nature Of the support.

Mesoporous silicas synthesis and application for lignin peroxidase immobilization by covalent binding method

Immobilization of enzymes on mesoporous silicas （MS） allows for good reusability. MS with two-dimensional hexagonal pores in diameter up to 14.13 nm were synthesized using Pluronic P123 as template and 1,3,5-triisopropylbenzene as a swelling agent in acetate buffer. The surface of MS was modified by the silanization reagents 3-aminopropyltriethoxysilane. Lignin peroxidase （LIP） was successfully immobilized on the modified MS through covalent binding method by four agents： glutaraldehyde, 1,4- phenylene diisothiocyanate, cyanotic chloride and water-soluble carbodiimide. Results showed that cyanotic chloride provided the best performance for LiP immobilization. The loaded protein concentration was 12.15 mg/g and the immobilized LiP activity was 812.9 U/L. Immobilized LiP had better pH stability. Acid Orange II was used to examine the reusability of immobilized LiP, showing more than 50% of the dye was decolorized at the fifth cycle.

Effect of covalent-binding modes of osteogenic-related peptides with artificial carriers on their biological activities in vivo

Covalent binding between bioactive substances and materials in different ways can significantly improve the bone inductivity and biological activity of bone repair materials.However,there is a lack of systematic understanding of how these binding modes affect biological activities of the active substances.In this study,four kinds of functionalized Multi-walled carbon nanotubes(MWCNTs)were prepared,ensuring the same grafting rate of different functional groups.Subsequently,two kinds of osteogenic-related peptides,bone morphogenetic protein-2 mimicking peptides and osteogenic growth mimicking peptides,were covalently bound to functionalized MWCNTs,ensuring the same molar mass of peptides bound to different functionalized MWCNTs in this process.Then the same amount of functionalized MWC-NTs/Peptides composites were introduced into the scaffolds,and through the ectopic osteogenesis model in rats and calvarial defect model in rabbits,ectopic osteogenesis and bone repair ability of the composites were analyzed.Furthermore,the effects of different covalent binding modes on peptide-induced osteogenesis and bone repair were studied.The results showed that the negative influencing trend of different covalent binding modes of osteogenic-related peptides with artificial carriers on their biological activities was in the order as follows:amide binding(carboxyl)>silane coupling>dopamine bind-ing>amide binding(amino),whose mechanism might be mainly that the covalent binding of peptides with different functional groups resulted in different charges.We believe that the results of this study have important guiding significance for the research and development of bone repair materials covalently bound with bioactive substances.

In vitro effect of biologically active coating on endothelialisation of Nitinol coils designed for the closure of intracardiac shunt

Objective:To evaluate in vitro the effect of biologically active coating on endothelialisation of Nitinol coils designed for the closure of intracardiac shunt. Methods:Covalently binding procedure was performed with chemical vapor deposition (CVD). Heparin(200 IU/ml),r-hirudin(21 nmol/ml),or fibronectin(15 μg/ml),respectively, were bound to the basic coating(poly(amino-p-xylylene-co-p-xylylen)). In vitro tests were performed on five different coating groups(n=2 each):uncoated Nitinol, basic coating,heparin-,r-hirudin-,and fibronectin-coating. Human umbilical vein endothelial cells(HUVECs) were cultured in the presence of tested coils for 48 and 72 h. Adhesion of HUVECs were evaluated with light microscopy,and with confocal laser scanning microscopy after double-staining of vinculin,phalloidin, Ki67 and fibronectin. Results: After 48 and 72 h of incubation,except HUVECs around the basic coating showing abnormal morphology, HUVECs were able to grow next to all kinds of coils. By CLSM adhering cells were typically confined to maximal three secondary coil windings. HUVECs adhered best to the fibronectin coated coils,followed by the uncoated Nitinol. HUVECs barely adhered to the basic coating. The adhesion to heparin coating or r-hirudin coating was observed with various cell densities. Heparin coating seemed to support cell adhesion better than r-hirudin coating did. The HUVECs assembled fibronectin matrix on the fibronectin coating and uncoated Nitinol coil. Vinculin was expressed on both fibronectin coating and uncoated Nitinol as well, mostly diffused,but occasionally also in focal contacts. Ki67 expression was mainly noticed on the uncoated Nitinol coil and fibronectin coated coil,only occasionally on other materials. The densities and morphology of HUVECs, adhered to the correspohding well surfaces,were similar for all wells. The only exception was the well with the basic coating coil after three days of incubation. In this case, the cell density was diminished,and the cells became more elongated. The cells grown on the well surfaces exhibited a more profound matrix assembly and more pronounced focal adhesions than the cells adhered to the coil surfaces did. Conclusion:Except for basic coating,biologically active substance coated or uncoated coils show no acute cytotoxicity on HUVECs. Due to better cell proliferation,cell matrix assembling,and focal adhesion,only fibronectin coating improves the endothelialisation of Nitinol coils.

Covalence bridge atomically precise metal nanocluster and metalorganic frameworks for enhanced photostability and photocatalysis

Metal nanoclusters(NCs)with precise structure and ultrasmall size have attracted great interests in catalysis.However,the poor stability has limited its large-scale use.Herein,we proposed the“covalence bridge”strategy to effectively connect atomically precise metal NCs and metal-organic frameworks.Benefiting from the covalent linkage,the synthesized UiO-66-NH2-Au25(LCys)18 showed outstanding stability after 16 h photocatalysis.Moreover,the covalence bridge created a strong metal-support interaction between the two components and provided an effective charge transport channel and thereby enhanced photocatalytic activity.UiO-66-NH2-Au25(L-Cys)18 displayed an exceptional photocatalytic H2 production rate,which is 21 and 90 times higher than that of UiO-66-NH2/Au25(PET)18(made by physically combination)and bare UiO-66-NH2,respectively.Thermodynamic and kinetic studies demonstrated that UiO-66-NH2-Au25(L-Cys)18 exhibited higher charge transfer efficiency,lower overpotential of water reduction and activation energy barrier compared with its counterparts.

In vitro and in vivo evaluation of implantable bacterial-killing coatings based on host defense peptides and their synthetic mimics

Contact-killing antimicrobial coatings based on host defense peptides(HDPs) and their synthetic mimics have shown potential as powerful tools to combat implant-associated infections. Covalent modification of the antimicrobial surface has been utilized to prevent early-stage microbial infections owing to the less drug-leaching possibility that is beneficial to human health and the natural environment. Although considerable progress has been achieved in preparing contact-killing antimicrobial surfaces, discussions focusing on the in vitro and in vivo evaluations of these surfaces are limited. In this review, we summarized the established in vitro methods to simulate the practical interaction of microbes with the surrounding biological environment and the reported in vivo studies at different implant sites. We suggested that the in vivo specific site infection model is essential to gain a comprehensive understanding of these antimicrobial coatings in the preclinical stage, which can be established based on investigations performed using various in vitro assays and conventional non-specific site infection models. Overall, these precedent studies focusing on bacterial contact-killing coatings modified with HDPs and HDP mimics can be considered as critical to assess the surface antibacterial ability and to guide the future developments and applications of antimicrobial surfaces.