Spectrophotometric Determination of Protein Concentration

Simonian, Michael H.

doi:10.1002/0471140856.txa03gs21

Cited by 17 publications

(16 citation statements)

References 6 publications

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“…Although the maximum absorbance of a protein actually occurs closer to 190 nm, A 205 has been favored in part due to the technical limitations of measuring at lower wavelengths . Still, whereas most common buffers and solutions in biological research are essentially transparent at 280 nm, many solutes will exhibit some absorbance at 205 nm (although this effect is even more pronounced at lower wavelengths) . Thus, one must consider, and carefully control for, any A 205 stemming from the buffer alone, as described below.…”

Section: Introductionmentioning

confidence: 99%

Sequence‐specific determination of protein and peptide concentrations by absorbance at 205 nm

2013

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Quantitative studies in molecular and structural biology generally require accurate and precise determination of protein concentrations, preferably via a method that is both quick and straightforward to perform. The measurement of ultraviolet absorbance at 280 nm has proven especially useful, since the molar absorptivity (extinction coefficient) at 280 nm can be predicted directly from a protein sequence. This method, however, is only applicable to proteins that contain tryptophan or tyrosine residues. Absorbance at 205 nm, among other wavelengths, has been used as an alternative, although generally using absorptivity values that have to be uniquely calibrated for each protein, or otherwise only roughly estimated. Here, we propose and validate a method for predicting the molar absorptivity of a protein or peptide at 205 nm directly from its amino acid sequence, allowing one to accurately determine the concentrations of proteins that do not contain tyrosine or tryptophan residues. This method is simple to implement, requires no calibration, and should be suitable for a wide range of proteins and peptides.

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Section: Introductionmentioning

confidence: 99%

Sequence‐specific determination of protein and peptide concentrations by absorbance at 205 nm

2013

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“…Tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) with aromatic side chains can also absorb light in this region and near (240–300 nm) UV region (Schmid, ). So, the characteristic absorption at 264 nm is mostly attributed to the integrated influence of Phe, Trp, and Tyr (Simonian, ). In this work, the results showed the absorbance intensity of SMP treated by sonication pretreatments was more strengthened than the control.…”

Section: Resultsmentioning

confidence: 99%

Ultrasound pretreatment of sunflower protein: Impact on enzymolysis, ACE‐inhibition activity, and structure characterization

Dabbour

Mintah

et al. 2020

J Food Process Preserv

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Impacts of dual‐frequency ultrasonic (DFU) with various operating modes (simultaneous and sequential modes) on degree of hydrolysis (DH) and conversion rate (CR) of sunflower meal protein (SMP) and activity of ACE‐inhibition of SMP hydrolysates were examined. Results revealed that sequential and simultaneous modes enhanced DH by 7.73% and 11.22%, and CR by 26.23% and 26.98% of SMP (p < .05). Additionally, the maximal ACE‐inhibition activity obtained at 75 min of enzymolysis was 64.46% for sonicated SMP hydrolysate (simultaneous mode), which was 32.52% higher than the control. Alterations in surface hydrophobicity, UV‐Vis spectra, and intrinsic fluorescence indicated unfolding of SMP. AFM showed that sonication decreased the particles size and altered the structure of SMP. Amino acids composition demonstrated that both DFU modes pretreatments could improve the hydrophobic amino acids. Consequently, sonication enhanced DH, CR of SMP, and the activity of ACE‐inhibition of hydrolysates confirmed by changes in SMP structure. Practical applications Sunflower protein, an extract from sunflower meal, is a potential source for preparing the bioactive compounds such as ACE‐ inhibition peptides. These peptides are safe, easy to absorb and stable, and have certain benefits for patients. Sonication with different operating frequency modes were applied to enhance enzymolysis of sunflower protein and ACE‐inhibition activity of its hydrolysates. Sonication was displayed to be an operative technique to yield hydrolysates with good ACE‐inhibition activity confirmed by monitoring the alterations in sunflower protein structure. Thus, sonication pretreatment of sunflower protein maybe practicable in the enzymolysis and the activity of ACE‐inhibition in industry.

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“…The molecular mass and peptide purity were confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) on an Ultraflex III spectrometer. For all assays, synthetic peptide concentrations were determined by using an Ultrospec 2100 spectrometer based on the extinction coefficient at 280 nm measured in water (36).…”

Section: Methodsmentioning

confidence: 99%

An Immunomodulatory Peptide Confers Protection in an Experimental Candidemia Murine Model

Freitas

Lima

Freire

et al. 2017

Antimicrob Agents Chemother

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Fungal Candida species are commensals present in the mammalian skin and mucous membranes. Candida spp. are capable of breaching the epithelial barrier of immunocompromised patients with neutrophil and cell-mediated immune dysfunctions and can also disseminate to multiple organs through the bloodstream. Here we examined the action of innate defense regulator 1018 (IDR-1018), a 12-amino-acid-residue peptide derived from bovine bactenecin (Bac2A): IDR-1018 showed weak antifungal and antibiofilm activity against a Candida albicans laboratory strain (ATCC 10231) and a clinical isolate (CI) (MICs of 32 and 64 g · ml Ϫ1 , respectively), while 8-fold lower concentrations led to dissolution of the fungal cells from preformed biofilms. IDR-1018 at 128 g · ml Ϫ1 was not hemolytic when tested against murine red blood cells and also has not shown a cytotoxic effect on murine monocyte RAW 264.7 and primary murine macrophage cells at the tested concentrations. IDR-1018 modulated the cytokine profile during challenge of murine bone marrow-derived macrophages with heatkilled C. albicans (HKCA) antigens by increasing monocyte chemoattractant protein 1 (MCP-1) and interleukin-10 (IL-10) levels, while suppressing tumor necrosis factor alpha (TNF-␣), IL-1␤, IL-6, and IL-12 levels. Mice treated with IDR-1018 at 10 mg · kg Ϫ1 of body weight had an increased survival rate in the candidemia model compared with phosphate-buffered saline (PBS)-treated mice, together with a diminished kidney fungal burden. Thus, IDR-1018 was able to protect against murine experimental candidemia and has the potential as an adjunctive therapy.

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Spectrophotometric Determination of Protein Concentration

Cited by 17 publications

References 6 publications

Sequence‐specific determination of protein and peptide concentrations by absorbance at 205 nm

Sequence‐specific determination of protein and peptide concentrations by absorbance at 205 nm

Ultrasound pretreatment of sunflower protein: Impact on enzymolysis, ACE‐inhibition activity, and structure characterization

An Immunomodulatory Peptide Confers Protection in an Experimental Candidemia Murine Model

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