Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins

Georgiou, Christos D.; Grintzalis, Konstantinos; Zervoudakis, George; Papapostolou, Ioannis

doi:10.1007/s00216-008-1996-x

Cited by 184 publications

(118 citation statements)

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“…1). It was similar to the spectrum of CBBG under pH=1, and its maximum absorbance was around 650 nm (Georgiou et al 2008). Under the conditions of testing, CEL, lignosulfonate, and alkali lignin were also neutral (Fig.…”

Section: Isothermal Titration Calorimetry and Ft-ir Analysis Of Combisupporting

confidence: 62%

Development of Method to Determine the Concentration of Alkali-Soluble Lignin using Coomassie Brilliant Blue G-250

Wang

Huang

2016

BioResources

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A new method involving the non-covalent binding of Coomassie Brilliant Blue G-250 (CBBG) to alkali-soluble lignin was developed. The binding of the dye to alkali-soluble lignin caused an increase in visible absorption at the maximum wavelength of 630 nm or 640 nm. A linear correlation of the absorbance at their maximum absorbing peak with alkali-soluble lignin concentration was observed. Lignin estimation in black liquor showed that the result of the new method and the gravimetric methods after acidification were closer to quantitative information than that obtained from UV spectroscopy. The isothermal titration calorimetric experiments, and Fourier Transform Infrared (FTIR) spectroscopy comparative analysis of precipitates washed by water, 4% ethanol, and 95% ethanol indicated that CBBG was bound to alkali-soluble lignin, and the binding was noncovalent. This potential method is reproducible, rapid, and cheap, and there is little or no inference from carbohydrate degradation products.

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Section: Isothermal Titration Calorimetry and Ft-ir Analysis Of Combisupporting

confidence: 62%

Development of Method to Determine the Concentration of Alkali-Soluble Lignin using Coomassie Brilliant Blue G-250

Wang

Huang

2016

BioResources

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“…17 The Coomassie blue method was used to determine the protein content. 18 Next, the percentage of entrapment was calculated. 19 The effect of temperature on the UOXLVs and free uricase was investigated in the temperature range of 20°C-70°C, and the effect of pH on the UOXLVs and free uricase was investigated in the pH range of 6.5-9.5 in borate buffers.…”

Section: Methods Preparation and Characteristics Of Uoxlvsmentioning

confidence: 99%

Improved biological properties and hypouricemic effects of uricase from Candida utilis loaded in novel alkaline enzymosomes

Tan¹,

Zhang²,

Wang³

et al. 2012

IJN

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Objective: Previous studies on various enzymosomes (functional lipid vesicles encapsulating an enzyme) have been mostly carried out in vitro and have focused on preserving catalytic activity and improving the stability of the enzyme. Until now, few studies have focused on their in vivo fate. Similarly, although we have previously reported the increased in vitro uricolytic activity (about 2.2 times higher than that of free uricase, or three times higher than that of PEGylated uricase, Puricase ® , under physiological pH and temperature) and improved stability of the novel alkaline enzymosomes (functional lipid vesicles encapsulating uricase from Candida utilis: uricase-containing lipid vesicles, UOXLVs), it is still necessary to study the biological properties and hypouricemic effects of UOXLVs in vivo. Methods:The enzyme kinetics, pharmacokinetics, pharmacodynamics, immunogenicity, and preliminary safety of UOXLVs were evaluated. Results: The Michaelis constant (K m ) value of the UOXLVs was slightly lower than that of the free enzyme. The enzyme release from the UOXLVs lasted over 12 hours and their circulation half-life was about sevenfold longer than that of the free uricase. Meanwhile, the UOXLVs had a 22-fold increase in the area under the curve compared with the free uricase. Furthermore, it took less than 3 hours for the UOXLVs to lower the plasma uric acid concentration from a high to a normal level, compared with 6 hours for the free uricase. In addition, the UOXLVs had much less immunogenicity than free uricase and were well tolerated by all animals throughout the observation period. Conclusion:The UOXLVs markedly improved the biological properties and enhanced the hypouricemic effects of uricase in vivo.

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“…The protease inhibitors PMSE/EDTA and the lipid antioxidant BHA were added in the phosphate buffer to protect the antioxidant enzymes and eliminate the artificial auto-oxidation of the fungal lipids, respectively, during homogenization. The mycelial homogenate was centrifuged at 20,000 ϫ g at 4°C for 15 min, the clear supernatant was collected, and its protein concentration was determined as previously described (28). This supernatant was used to determine lipid peroxidation, polyphenols, certain thiol redox state parameters, and also the activity of certain antioxidant enzymes.…”

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confidence: 99%

“…Nonetheless, ammonium sulfate does not interfere with the activities of the assayed enzymes; commercial preparations of superoxide dismutase, catalase, and glutathione reductase from various sources are actually stabilized as ammonium sulfate suspensions, whereas the purification of glutathione peroxidase involves ammonium sulfate fractionation. Enzyme activities were expressed per protein, the concentration of which was determined as previously described (28). The SOD specific activity was assayed as previously reported (36) and expressed as SOD units per mg of protein (using a standard curve made with pure bovine erythrocyte SOD).…”

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confidence: 99%

Role of Oxidative Stress in Sclerotial Differentiation and Aflatoxin B1 Biosynthesis in Aspergillus flavus

Grintzalis

Vernardis

Klapa

et al. 2014

Appl Environ Microbiol

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104

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cWe show here that oxidative stress is involved in both sclerotial differentiation (SD) and aflatoxin B1 biosynthesis in Aspergillus flavus. Specifically, we observed that (i) oxidative stress regulates SD, as implied by its inhibition by antioxidant modulators of reactive oxygen species and thiol redox state, and that (ii) aflatoxin B1 biosynthesis and SD are comodulated by oxidative stress. However, aflatoxin B1 biosynthesis is inhibited by lower stress levels compared to SD, as shown by comparison to undifferentiated A. flavus. These same oxidative stress levels also characterize a mutant A. flavus strain, lacking the global regulatory gene veA. This mutant is unable to produce sclerotia and aflatoxin B1. (iii) Further, we show that hydrogen peroxide is the main modulator of A. flavus SD, as shown by its inhibition by both an irreversible inhibitor of catalase activity and a mimetic of superoxide dismutase activity. On the other hand, aflatoxin B1 biosynthesis is controlled by a wider array of oxidative stress factors, such as lipid hydroperoxide, superoxide, and hydroxyl and thiyl radicals. Humans and animals are exposed to carcinogenic aflatoxins through contaminated food and feed, air, and drinking water (1, 2). Aspergillus flavus is the primary cause of aflatoxin-contaminated crops. A. flavus is a heterothallic fungus, and laboratory crosses produce ascospore-bearing ascocarps embedded within sclerotia. In the field, sclerotia are dispersed during crop harvest and require an additional incubation period on the soil for sexual reproduction (3). Despite the significant contribution of A. flavus to crop aflatoxin contamination, it is not yet known what the role of oxidative stress is for its sclerotial differentiation (SD) and aflatoxin B1 biosynthesis. Deciphering this relationship could contribute to the development of nontoxic antifungal means via the coinhibition of A. flavus SD and aflatoxin B1 biosynthesis.Several toxigenic and phytopathogenic fungi spread and survive in nature through the formation of conidiophores and resistant sclerotia. It has been known that oxidative stress regulates the sclerotial differentiation of filamentous phytopathogenic fungi such as Rhizoctonia solani, Sclerotium rolfsii, Sclerotinia sclerotiorum, and Sclerotinia minor (4, 5). Moreover, it has been established that the regulation of morphogenesis in aspergilli and other fungi is genetically linked to secondary metabolism (6-9). In A. flavus, both SD and aflatoxin biosynthesis are governed by the regulatory protein VeA (10). Deletion of the veA gene in this fungus results in the inhibition of sclerotia formation and aflatoxin biosynthesis (10). However, it is not known whether SD in A. flavus is regulated by oxidative stress and whether the deletion of veA could alter its oxidative stress levels.Previous reports have linked aflatoxin biosynthesis with oxidative stress in A. flavus and A. parasiticus both at the metabolic and transcriptional levels. Specifically, aflatoxin biosynthesis in both species is activated by high ...

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Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins

Cited by 184 publications

References 10 publications

Development of Method to Determine the Concentration of Alkali-Soluble Lignin using Coomassie Brilliant Blue G-250

Development of Method to Determine the Concentration of Alkali-Soluble Lignin using Coomassie Brilliant Blue G-250

Improved biological properties and hypouricemic effects of uricase from Candida utilis loaded in novel alkaline enzymosomes

Role of Oxidative Stress in Sclerotial Differentiation and Aflatoxin B1 Biosynthesis in Aspergillus flavus

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