Quantitative serine protease assays based on formation of copper(<scp>ii</scp>)–oligopeptide complexes

Ding, Xiaokang; Yang, Kun‐Lin

doi:10.1039/c4an01731e

Cited by 17 publications

(18 citation statements)

References 33 publications

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“…For example, amino terminal copper and nickel (ATCUN)-binding peptide with a structural characterization comprising of a free NH 2 -terminus, a histidine (His) residue in the third position and two intervening peptide nitrogens (denoted as NH 2 -X-X-His sequence) exhibits high affinity to Cu 2+ ion. [37][38][39][40] The formation of ATCUN-Cu 2+ complex depresses Cu 2+ -initiated redox cycling for AA oxidation. 40 In this work, we demonstrated that the PSA substrate peptide (DAHSSKLQLAPP) that contains an ATCUN motif of DAH can sequestrate Cu 2+ by the formation of ATCUN-Cu 2+ complex ( Figure 1A), thus depressing the Cu 2+ -catalyzed oxidation of AA.…”

Section: Introductionmentioning

confidence: 99%

Gold nanoparticle-based colorimetric method for the detection of prostate-specific antigen

Xia¹,

Deng²,

Wang³

et al. 2018

IJN

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BackgroundProstate-specific antigen (PSA), a serine protease, is a biomarker for preoperative diagnosis and screening of prostate cancer and monitoring of its posttreatment.MethodsIn this work, we reported a colorimetric method for clinical detection of PSA using gold nanoparticles (AuNPs) as the reporters. The method is based on ascorbic acid (AA)-induced in situ formation of AuNPs and Cu2+-catalyzed oxidation of AA. Specifically, HAuCl4 can be reduced into AuNPs by AA; Cu2+ ion can catalyze the oxidation of AA by O2 to inhibit the formation of AuNPs. In the presence of the PSA-specific peptide (DAHSSKLQLAPP)-modified gold-coated magnetic microbeads (MMBs; denoted as DAHSSKLQLAPP-MMBs), complexation of Cu2+ by the MMBs through the DAH–Cu2+ interaction depressed the catalyzed oxidation of AA and thus allowed for the formation of red AuNPs. However, once the peptide immobilized on the MMB surface was cleaved by PSA, the DAHSSKLQ segment would be released. The resultant LAPP fragment remaining on the MMB surface could not sequestrate Cu2+ to depress its catalytic activity toward AA oxidation. Consequently, no or less AuNPs were generated.ResultsThe linear range for PSA detection was found to be 0~0.8 ng/mL with a detection limit of 0.02 ng/mL. Because of the separation of cleavage step and measurement step, the interference of matrix components in biological samples was avoided.ConclusionThe high extinction coefficient of AuNPs facilitates the colorimetric analysis of PSA in serum samples. This work is helpful for designing of other protease biosensors by matching specific peptide substrates.

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Section: Introductionmentioning

confidence: 99%

Gold nanoparticle-based colorimetric method for the detection of prostate-specific antigen

Xia¹,

Deng²,

Wang³

et al. 2018

IJN

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“…MTS reagents are a reasonable choice to suppress irreversible denaturation of globular proteins triggered by disulphide bond breakage due to their high thiol-specificity and rapid protection ability. Traditionally, copper(II) ions were used as an effective suppressor of disulphide-thiol exchange protein denaturation 14 , 15 , but copper(II) catalyses unfavourable methionine oxidation, and the formation of copper(II) hydroxide precipitates often interferes with optical analysis 29 . Thiol-specific irreversible blockers, such as NEM and IAA, protect thiols generated by breakage of disulphide bonds upon heating by S -alkylation.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of irreversible protein thermal inactivation caused by breakage of disulphide bonds using methanethiosulphonate

Futami

Miyamoto

Hagimoto

et al. 2017

Sci Rep

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Many extracellular globular proteins have evolved to possess disulphide bonds in their native conformations, which aids in thermodynamic stabilisation. However, disulphide bond breakage by heating leads to irreversible protein denaturation through disulphide-thiol exchange reactions. In this study, we demonstrate that methanethiosulphonate (MTS) specifically suppresses the heat-induced disulphide-thiol exchange reaction, thus improving the heat-resistance of proteins. In the presence of MTS, small globular proteins that contain disulphides can spontaneously refold from heat-denatured states, maintaining wild-type disulphide pairing. Because the disulphide-thiol exchange reaction is triggered by the generation of catalytic amounts of perthiol or thiol, rapid and specific perthiol/thiol protection by MTS reagents prevents irreversible denaturation. Combining MTS reagents with another additive that suppresses chemical modifications, glycinamide, further enhanced protein stabilisation. In the presence of these additives, reliable remnant activities were observed even after autoclaving. However, immunoglobulin G and biotin-binding protein, which are both composed of tetrameric quaternary structures, failed to refold from heat-denatured states, presumably due to chaperon requirements. Elucidation of the chemical modifications involved in irreversible thermoinactivation is useful for the development of preservation buffers with optimum constitutions for specific proteins. In addition, the impact of disulphide bond breakage on the thermoinactivation of proteins can be evaluated using MTS reagents.

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“…Although some of the methods described here were not originally designed to screen the activity of viral proteases in the presence of inhibitors, they can be adapted for this purpose by simply changing the peptide sequence to the target site of the protease of interest. The cleavage yield is usually monitored either colorimetrically (Ding and Yang, 2015;Zhou et al, 2014) or as a change in fluorescence triggered by the release of fluorescent labels such as 7-amido-4-methylcoumarin (AMC) or rhodamine (Grant et al, 2002). Fluorogenic substrates have been used to determine the activity of coronavirus proteases and screen inhibitors (Kuo et al, 2004;Lee et al, 2014;Park et al, 2017;Song et al, 2014;Tomar et al, 2015;Wang et al, 2016;Yang et al, 2005;Zhao et al, 2008).…”

Section: Assay and Methods For Screening And Evaluating Viral Maturatmentioning

confidence: 99%

In vitro methods for testing antiviral drugs

Rumlová

Ruml

2018

Biotechnology Advances

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Despite successful vaccination programs and effective treatments for some viral infections, humans are still losing the battle with viruses. Persisting human pandemics, emerging and re-emerging viruses, and evolution of drug-resistant strains impose continuous search for new antiviral drugs. A combination of detailed information about the molecular organization of viruses and progress in molecular biology and computer technologies has enabled rational antivirals design. Initial step in establishing efficacy of new antivirals is based on simple methods assessing inhibition of the intended target. We provide here an overview of biochemical and cell-based assays evaluating the activity of inhibitors of clinically important viruses.

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Quantitative serine protease assays based on formation of copper(ii)–oligopeptide complexes

Cited by 17 publications

References 33 publications

Gold nanoparticle-based colorimetric method for the detection of prostate-specific antigen

Gold nanoparticle-based colorimetric method for the detection of prostate-specific antigen

Evaluation of irreversible protein thermal inactivation caused by breakage of disulphide bonds using methanethiosulphonate

In vitro methods for testing antiviral drugs

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