Isoelectric point of free and adsorbed cytochrome c determined by various methods

Hristova, Svetlana H.; Zhivkov, Alexandar M.

doi:10.1016/j.colsurfb.2018.10.080

Cited by 24 publications

(23 citation statements)

References 52 publications

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“…Another IEP measurement is isoelectric focusing (IEF) [ 13 ] and capillary isoelectric focusing (CIEF) [ 14 ]. Both IEF methods require the fluorescent tagging of viruses, which requires pure, concentrated solutions [ 15 ]. Different methods are needed to measure virus IEP in natural solutions without high purity and concentration requirements.…”

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confidence: 99%

Single-Particle Characterization of SARS-CoV-2 Isoelectric Point and Comparison to Variants of Interest

et al. 2021

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SARS-CoV-2, the cause of COVID-19, is a new, highly pathogenic coronavirus, which is the third coronavirus to emerge in the past 2 decades and the first to become a global pandemic. The virus has demonstrated itself to be extremely transmissible and deadly. Recent data suggest that a targeted approach is key to mitigating infectivity. Due to the proliferation of cataloged protein and nucleic acid sequences in databases, the function of the nucleic acid, and genetic encoded proteins, we make predictions by simply aligning sequences and exploring their homology. Thus, similar amino acid sequences in a protein usually confer similar biochemical function, even from distal or unrelated organisms. To understand viral transmission and adhesion, it is key to elucidate the structural, surface, and functional properties of each viral protein. This is typically first modeled in highly pathogenic species by exploring folding, hydrophobicity, and isoelectric point (IEP). Recent evidence from viral RNA sequence modeling and protein crystals have been inadequate, which prevent full understanding of the IEP and other viral properties of SARS-CoV-2. We have thus experimentally determined the IEP of SARS-CoV-2. Our findings suggest that for enveloped viruses, such as SARS-CoV-2, estimates of IEP by the amino acid sequence alone may be unreliable. We compared the experimental IEP of SARS-CoV-2 to variants of interest (VOIs) using their amino acid sequence, thus providing a qualitative comparison of the IEP of VOIs.

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confidence: 99%

Single-Particle Characterization of SARS-CoV-2 Isoelectric Point and Comparison to Variants of Interest

et al. 2021

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“…Polyelectrolyte nanogels have been attested to be suitable enzyme carriers in previous studies. , Therefore, the obtained PAA nanogels with negative charges are supposed to load positive enzyme via electrostatic attraction. We select PAA nanogels prepared with 20% DHEA and cytochrome c (cyt c , pI 10.5, M w 12.5 kDa, ∼3.4 nm) as the model enzyme. pH was controlled at 6.5 which gives sufficient negative and positive charges of nanogels and cyt c, respectively (Figure S4).…”

Section: Resultsmentioning

confidence: 99%

Synthesis of Anionic Nanogels for Selective and Efficient Enzyme Encapsulation

Wan

Cai

et al. 2022

Langmuir

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Polyelectrolyte nanogels containing cross-linked ionic polymer networks feature both soft environment and intrinsic charges which are of great potential for enzyme encapsulation. In this work, well-defined poly(acrylic acid) (PAA) nanogels have been synthesized based on a facile strategy, namely, electrostatic assembly directed polymerization (EADP). Specifically, AA monomers are polymerized together with a crosslinker in the presence of a cationic-neutral diblock copolymer as the template. Effects of control factors including pH, salt concentration, and cross-linking degree have been investigated systematically, based on which the optimal preparation of PAA nanogels has been established. The obtained nanogel features not only compatible pocket for safely loading enzymes without disturbing their structures, but also abundant negative charges which enable selective and efficient encapsulation of cationic enzymes. The loading capacities of PAA nanogels for cytochrome (cyt c) and lysozyme are 100 and 125 μg/mg (enzyme/nanogel), respectively. More notably, the PAA network seems to modulate a favorable microenvironment for cyt c and induces 2-fold enhanced activity for the encapsulated enzymes, as indicated by the steady-state kinetic assay. Our study reveals the control factors of EADP for optimal synthesis of anionic nanogels and validates their distinctive advances with respect to efficient loading and activation of cationic enzymes.

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“…The cytC–MM suspension was prepared at pH 6 (at which the cytC globules are positively charged [ 7 , 8 ], which is ensured to condition their electrostatic adsorption on the negatively charged MM nanoplates) under continuous stirring for 30 min at room temperature by slow addition of the protein solution to the MM suspension to a final concentration of 10 mg/L cytC in 3 mg/L MM, at which ratio protein adsorption was saturated [ 14 ]). The pH of the suspension was slightly adjusted to pH 6.0 by the addition of a very small volume of concentrated KOH.…”

Section: Methodsmentioning

confidence: 99%

“…In order for the electrostatic adsorption of cytC to take place, its charge must be opposite to that of the colloid particles. As in other proteins, the charge of cytC is pH-dependent due to the ionization of the chargeable amino acid residues; the ratio of the carboxylic (COO – ) and amino (NH 3 + ) groups determines its isoelectric point, which emerges at pH 9 [ 7 , 8 ]. Since the cytC globules are positively charged at neutral pH (pH 6 is the most suitable because a buffer is not necessary), we have chosen montmorillonite (MM, a laminar alumosilicate clay mineral) whose charge is negative and pH-independent due to the isomorphic exchange of Al 3+ in the central sublayer with extrinsic bivalent metal ions with lower valency (Zn 2+ and Fe 2+ ).…”

Section: Introductionmentioning

confidence: 99%

Protein–Mineral Composite Particles with Logarithmic Dependence of Anticancer Cytotoxicity on Concentration of Montmorillonite Nanoplates with Adsorbed Cytochrome c

Hristova

Zhivkov

2023

Pharmaceutics

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Montmorillonite (MM) colloid nanoplates have high adsorption capacity due to their large size/thickness ratio, which allows them to be used as carriers for drug delivery. Upon adsorption of the mitochondrial protein cytochrome c (cytC) onto MM plates, the composite cytC–MM particles acquire anticancer properties because of the ability of cancer cells to phagocytize submicron particles (in contrast to the normal cells). In this way, exogenous cytC can be introduced into tumor cells, thereby triggering apoptosis—an irreversible cascade of biochemical reactions leading to cell death. In the present study, we investigated the physicochemical properties of cytC–MM particles as a function of the cytC concentration in the suspension, namely, the electrophoretic mobility, the mass increment of MM monoplates upon cytC adsorption, the ratio of the adsorbed to the free cytC in the bulk, the protein density on the MM’s surface, the number of cytC globules adsorbed on an MM monoplate, the concentration of cytC–MM composite particles in the suspension, and the dependence of cytotoxicity on the cytC–MM particle concentration. For this purpose, we used microelectrophoresis, static and electric light scattering, and a colon cancer cell culture to test the cytotoxic effects of the cytC–MM suspensions. The results show that the cytotoxicity depends linearly on the logarithm of the particle concentration in the cytC–MM suspension reaching 97%.

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Isoelectric point of free and adsorbed cytochrome c determined by various methods

Cited by 24 publications

References 52 publications

Single-Particle Characterization of SARS-CoV-2 Isoelectric Point and Comparison to Variants of Interest

Single-Particle Characterization of SARS-CoV-2 Isoelectric Point and Comparison to Variants of Interest

Synthesis of Anionic Nanogels for Selective and Efficient Enzyme Encapsulation

Protein–Mineral Composite Particles with Logarithmic Dependence of Anticancer Cytotoxicity on Concentration of Montmorillonite Nanoplates with Adsorbed Cytochrome c

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