“…e involvement of dynamic quenching in the interaction of BSA and 5d was not considered as the absorption spectrum of BSA in presence of 5d might have been una ected in this case. However, an increase in the absorption intensity of BSA on its interaction with 5d suggests complex formation between them ( Figure 5(b)) and involvement of static quenching [43]. Moreover, the no-overlap was observed in the absorption spectra of BSA, 5d, BSA-5d, and (BSA-5d)-5d.…”
Section: Uv-vismentioning
confidence: 95%
“…ϵ(λ) denotes molar absorptivity coe cient of acceptor. e value for K 2 refractive index of the medium was taken as 1.336 and ϕ D � 0.118 for BSA [43,44]. e calculated values for E, R 0 , and J(λ) are presented in Table 4.…”
Section: Energy Transfermentioning
confidence: 99%
“…e 3D fluorescence spectroscopy has gained quite popularity to access the protein structural changes. It gives a detailed account of the conformation modifications in protein due to protein-ligand interaction [21,43].…”
In this research, the pyrazoline pyridazine derivative 7-methyl-2-phenyl-4-(3,4,5-trimethoxyphenyl)-2H-pyrazolo[3,4-d]pyridazine (5d) was studied for its interaction with bovine serum albumin (BSA). Various spectroscopic techniques along with molecular docking analysis were utilized to understand the mechanism of interaction. The quenching of BSA fluorescence by using investigational drug 5d was the basic principle for the methodology. Spectrofluorometric methods and UV-absorption studies were conducted for exploration of the 5d and BSA binding mechanism. The fluorescence quenching mechanism involved in BSA and 5d interaction was static quenching, and a complex formation also occurred between them. Both enthalpy and entropy attained positive values suggesting involvement of hydrophobic forces in BSA and 5d interaction. The Förster distance of 2.23 nm was calculated by fluorescence resonance energy transfer (FRET). An alteration in BSA secondary structure was proven from the conformational studies of BSA-5d interaction. This binding interaction study provided a basis to comprehend the binding interaction between 5d and BSA. These results provided information about sites of BSA involved in its interaction with 5d.
“…e involvement of dynamic quenching in the interaction of BSA and 5d was not considered as the absorption spectrum of BSA in presence of 5d might have been una ected in this case. However, an increase in the absorption intensity of BSA on its interaction with 5d suggests complex formation between them ( Figure 5(b)) and involvement of static quenching [43]. Moreover, the no-overlap was observed in the absorption spectra of BSA, 5d, BSA-5d, and (BSA-5d)-5d.…”
Section: Uv-vismentioning
confidence: 95%
“…ϵ(λ) denotes molar absorptivity coe cient of acceptor. e value for K 2 refractive index of the medium was taken as 1.336 and ϕ D � 0.118 for BSA [43,44]. e calculated values for E, R 0 , and J(λ) are presented in Table 4.…”
Section: Energy Transfermentioning
confidence: 99%
“…e 3D fluorescence spectroscopy has gained quite popularity to access the protein structural changes. It gives a detailed account of the conformation modifications in protein due to protein-ligand interaction [21,43].…”
In this research, the pyrazoline pyridazine derivative 7-methyl-2-phenyl-4-(3,4,5-trimethoxyphenyl)-2H-pyrazolo[3,4-d]pyridazine (5d) was studied for its interaction with bovine serum albumin (BSA). Various spectroscopic techniques along with molecular docking analysis were utilized to understand the mechanism of interaction. The quenching of BSA fluorescence by using investigational drug 5d was the basic principle for the methodology. Spectrofluorometric methods and UV-absorption studies were conducted for exploration of the 5d and BSA binding mechanism. The fluorescence quenching mechanism involved in BSA and 5d interaction was static quenching, and a complex formation also occurred between them. Both enthalpy and entropy attained positive values suggesting involvement of hydrophobic forces in BSA and 5d interaction. The Förster distance of 2.23 nm was calculated by fluorescence resonance energy transfer (FRET). An alteration in BSA secondary structure was proven from the conformational studies of BSA-5d interaction. This binding interaction study provided a basis to comprehend the binding interaction between 5d and BSA. These results provided information about sites of BSA involved in its interaction with 5d.
“…It can be deduced that quenching efficacy is varied by temperature. To quantify the impact of temperature and identify the quenching mechanism, the Stern–Volmer Equation (1) was plotted.32 Figure 1Fluorescence quenching of CAT in the presence of different concentrations of SiO 2 NPs: 0 (red), 1 (blue), 5 (purple), 10 (grey), 15 (yellow) and 20 μM (green) at 298 ( A ), 310 ( B ) and 315 K ( C ).…”
Aim
Nanoparticles (NPs) have been receiving potential interests in protein delivery and cell therapy. As a matter of fact, NPs may be used as great candidates in promoting cell therapy by catalase (CAT) delivery into high oxidative stress tissues. However, for using NPs like SiO
2
as carriers, the interaction of NPs with proteins and mesenchymal stem cells (MSCs) should be explored in advance.
Methods
In the present study, the interaction of SiO
2
NPs with CAT and human MSCs (hMSCs) was explored by various spectroscopic methods (fluorescence, circular dichroism (CD), UV-visible), molecular docking and dynamics studies, and cellular (MTT, cellular morphology, cellular uptake, lactate dehydrogenase, ROS, caspase-3, flow cytometry) assays.
Results
Fluorescence study displayed that both dynamic and static quenching mechanisms and hydrophobic interactions are involved in the spontaneous interaction of SiO
2
NPs with CAT. CD spectra indicated that native structure of CAT remains stable after interaction with SiO
2
NPs. UV-visible study also revealed that the kinetic parameters of CAT such as
Km, Vmax, Kcat
, and enzyme efficiency were not changed after the addition of SiO
2
NPs. Molecular docking and dynamics studies showed that Si and SiO
2
clusters interact with hydrophobic residues of CAT and SiO
2
cluster causes minor changes in the CAT structure at a total simulation time of 200 ps. Cellular assays depicted that SiO
2
NPs induce significant cell mortality, change in cellular morphology, cellular internalization, ROS elevation, and apoptosis in hMSCs at higher concentration than 100 µg/mL (170 µM).
Conclusion
The current results suggest that low concentrations of SiO
2
NPs induce no substantial change or mortality against CAT and hMSCs, and potentially useful carriers in CAT delivery to hMSC.
“…Various reports have shown the utility of UV-vis absorption measurements for drug-protein interaction assays [14][15][16][17]. This approach was mainly aimed at further confirming the statically formed protein-drug complex and observing protein structural changes once it is bound to the ligand.…”
The interactions of novel anti-cancer therapeutic agents with the different plasma and tissue components, specifically serum albumins, have lately gained considerable attention due to the significant influence of such interactions on the pharmacokinetics and/or -dynamics of this important class of therapeutics. Nazartinib (EGF 816; NAZ) is a new anti-cancer candidate proposed as a third-generation epidermal growth factor receptor tyrosine kinase inhibitor that is being developed and clinically tested for the management of non-small cell lung cancer. The current study aimed to characterize the interaction between NAZ and human serum albumin (HSA) using experimental and theoretical approaches. Experimental results of fluorescence quenching of HSA induced by NAZ revealed the development of a statically formed complex between NAZ and HSA. Interpretation of the observed fluorescence data using Stern–Volmer, Lineweaver–Burk and double-log formulae resulted in binding constants for HSA-NAZ complex in the range of (2.34–2.81) × 10
4
M
–1
over the studied temperatures. These computed values were further used to elucidate thermodynamic attributes of the interaction, which showed that NAZ spontaneously binds to HSA with a postulated electrostatic force-driven interaction. This was further verified by theoretical examination of the NAZ docking on the HSA surface that revealed an HSA-NAZ complex where NAZ is bound to HSA Sudlow site I driven by hydrogen bonding in addition to electrostatic forces in the form of pi-H bond. The HSA binding pocket for NAZ was shown to encompass ARG 257, ARG 222, LYS 199 and GLU 292 with a total binding energy of −25.59 kJ mol
–1
.
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