Improving data quality and expanding BioSAXS experiments to low-molecular-weight and low-concentration protein samples

2020 ◽  
Vol 76 (10) ◽  
pp. 971-981
Author(s):  
Albert Castellví ◽  
Carlos Pascual-Izarra ◽  
Eva Crosas ◽  
Marc Malfois ◽  
Judith Juanhuix

The addition of compounds to scavenge the radical species produced during biological small-angle X-ray scattering (BioSAXS) experiments is a common strategy to reduce the effects of radiation damage and produce better quality data. As almost half of the experiments leading to structures deposited in the SASBDB database used scavengers, finding potent scavengers would be advantageous for many experiments. Here, four compounds, three nucleosides and one nitrogenous base, are presented which can act as very effective radical-scavenging additives and increase the critical dose by up to 20 times without altering the stability or reducing the contrast of the tested protein solutions. The efficacy of these scavengers is higher than those commonly used in the field to date, as verified for lysozyme solutions at various concentrations from 7.0 to 0.5 mg ml−1. The compounds are also very efficient at mitigating radiation damage to four proteins with molecular weights ranging from 7 to 240 kDa and pH values from 3 to 8, with the extreme case being catalase at 6.7 mg ml−1, with a scavenging factor exceeding 100. These scavengers can therefore be instrumental in expanding BioSAXS to low-molecular-weight and low-concentration protein samples that were previously inaccessible owing to poor data quality. It is also demonstrated that an increase in the critical dose in standard BioSAXS experiments leads to an increment in the retrieved information, in particular at higher angles, and thus to higher resolution of the model.

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jun Ma ◽  
Xiankun Zeng ◽  
Min Zhou ◽  
Le Cheng ◽  
Difeng Ren

AbstractSpirulina platensis protein hydrolysates were prepared by digesting protein extracts with papain, and the hydrolysates were separated into 30, 10, and 3 kDa weights using membrane ultrafiltration. The 0–3 kDa low-molecular-weight Spirulina peptides (LMWSPs) proved the highest chemical antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability, hydroxyl radical (·OH) scavenging activities and total antioxidant capacity. Cellular antioxidant ability of LMWPs fractions against 2000 μg/mL H2O2 induced oxidative damage of L02 cells were investigated. The MTT assay results displayed that LMWSPs at different concentrations (0–1000 μg/mL) had proliferation effect on the L02 cells and that treatment of the L02 cells with the 1000 μg/mL LMWSPs (0–3 kDa) significantly prevented H2O2-induced oxidative damage compared with control cells. Moreover, the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe assay showed that the levels of ROS and NO were significantly lower in the experimental group that was treated with the peptides for 24 h than in the control group. Furthermore, using the corresponding kits, the treatment inhibited the reduction of SOD activity and the increase of MDA contents in the L02 cells. Therefore, LMWSPs (0–3 kDa) may have potential applications in antioxidant and liver health products.


Author(s):  
E Podzorski ◽  
F E Wells

The effect of matrix albumin concentration on reduction of NBT by DMF has been investigated together with the effect of cationic additives. Decreased albumin concentration and increased cationic detergent concentration both increased the sensitivity of the assay due to kinetic effects, but addition of a low molecular weight cation had no effect. The reduction of NBT by glycated albumin was not increased in sensitivity by a protein matrix of low concentration. Due to the complexity and poorly understood nature of interaction between DMF and albumin in the reduction of NBT, the authors do not consider DMF to be a suitable primary calibrant for the fructosamine assay.


1996 ◽  
Vol 44 (4) ◽  
pp. 967-971 ◽  
Author(s):  
Juan Bautista ◽  
Inmaculada Hernandez-Pinzon ◽  
Manuel Alaiz ◽  
Juan Parrado ◽  
Francisco Millan

BioResources ◽  
2010 ◽  
Vol 6 (1) ◽  
pp. 232-242
Author(s):  
Chuan-Ling Si ◽  
Yi-Yuan Lu ◽  
Yu Zhang ◽  
Jie Xu ◽  
Pan-Pan Qin ◽  
...  

Triploid Populus tomentosa Carr. (Salicaceae) is a good alternative to meet the increasing need of the global pulp and paper industry. Meanwhile, the xylem of this species could be a useful bioresource to develop low molecular extractives with significant bioactive potential. In the present work, a phytochemical investigation on aqueous EtOH extractives of Triploid P. tomentosa xylem, by systematical performance of Sephadex LH-20 open column chromatography and Thin Layer Chromatography (TLC), resulted in the isolation of two phenolic acids (ρ-coumaric acid (I) and caffeic acid (II)), two flavonoids (apigenin (III) and luteolin (IV)), and three phenolic glucosides (salicortin (V), salireposide (VI) and populoside (VII)). The structure elucidation and determination of the isolated extractives were based on their spectroscopical data and physiochemical evidences. This was the first time to report the low molecular weight extractives of Triploid P. tomentosa. Various low molecular weight extractives fromTriploid P. tomentosa xylem exhibited significant antioxidative activities by DPPH and hydroxyl radical scavenging assays.


2019 ◽  
Author(s):  
Sushanta Kumar Barik ◽  
Keshar Kunja Mohanty ◽  
Deepa Bisht ◽  
Partha Sarathi Mohanty ◽  
Shripad Patil ◽  
...  

Abstract Human plasma contains high amount of abundant proteins like albumin and globulin. Normally, the proteins having potential for biomarkers are present in very low concentration in human plasma. To resolve the low concentration proteins in polyacrylamide gel, the removal of high abundant proteins from plasma are very essential. Polyethylene glycol is a nontoxic, water soluble synthetic polymer has several applications in chemical and biomedical industries. Various molecular variants of poly ethylene glycol is available and used in protein purification. The mechanism behind the use of high concentration of polyethylene glycol is it binds the molecule in more compact or interpenetrates forming a gel like network surrounding the molecule. Polyethylene glycol -6000 removes the high abundant proteins like Albumin and Globulin in the HIV -1 infected plasma samples and concentrates the low molecular weight proteins as the low molecular weight proteins are essential in biomarker study.


2016 ◽  
Vol 11 ◽  
pp. S61-S67 ◽  
Author(s):  
Jinpeng Wang ◽  
Lian Zhang ◽  
Zhengyu Jin

Low-molecular-weight chondroitin sulfate was obtained by degradation of chondroitin sulfate using hyaluronidase. Then, separated with sephadex G25, DEAE-52, and finally purified with AKATA superpeptide separation system and fluorescence-assisted carbohydrate electrophoresis. The main compo-nents detected by high performance gel-filtration chromatography were disaccharide, tetrasaccharide, hexasaccharide with molecular weight of 521, 1024 and 1527 Da, respectively. The anti-oxidant activity of these three oligosaccharides in vitro showed that the reducing power (maximum value at 10 mg/mL) and superoxide anion radical scavenging abilities were increased (maximum value at 4 mg/mL) with an increased in their concentration. There were no significant differences of the anti-oxidant properties between those three oligosaccharides. 


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