Abstract
Benzothiazole based cyanine dyes with bridged groups in the pentamethine chain were studied as potential far-red fluorescent probes for protein detection. Spectral-luminescent properties were characterized for unbound dyes and in the presence of serum albumins (bovine (BSA), human (HSA), equine (ESA)), and globular proteins (β-lactoglobulin, ovalbumin). We have observed that the addition of albumins leads to a significant increase in dyes fluorescence intensity. However, the fluorescent response of dyes in the presence of other globular proteins was notably lower. The value of fluorescence quantum yield for dye bearing a sulfonate group complexed with HSA amounted to 42% compared with 0.2% for the free dye. The detection limit of HSA by this dye was greater than 0.004 mg ml−1 which indicates the high sensitivity of dye to low HSA concentrations. Modelling of structure of the dyes complexes with albumin molecules was performed by molecular docking. According to these data, dyes could bind to up to five sites on the HSA molecule; the most preferable are the haemin-binding site in subdomain IB and the dye-binding site in the pocket between subdomains IA, IIA and IIIA. This work confirms that pentamethine cyanine dyes could be proposed as powerful far-red fluorescent probes applicable for highly sensitive detection of albumins.
Highlights
This article has been edited by the Royal Society of Chemistry, including the commissioning, peer review process and editorial aspects up to the point of acceptance
We have shown that squaraine dyes are efficient noncovalent labels for albumin detection, exhibiting high fluorescence quantum yields when bound to these proteins [14,15,16]. 2-Quinolone and coumarin polymethines were shown to be able to increase the fluorescence intensity one hundred times with bright emission in the presence of albumin [17]
We observed that all far-red dyes studied in this work exhibit a stronger fluorescent ‘light-up’ response in the presence of serum albumins (SAs) (BSA, human serum albumin (HSA) and equine serum albumin (ESA)) than in the presence of other globular proteins (OVA and BLG)
Summary
This article has been edited by the Royal Society of Chemistry, including the commissioning, peer review process and editorial aspects up to the point of acceptance. The bovine serum albumin (BSA) globule is known to be able to bind (at pH values between 5 and 10) five molecules of 1,8-ANS resulting in ANS fluorescent response [10,11] Another well-known dye for protein characterization is Nile Red, which can be applied for monitoring conformational changes of different proteins. Cyanine dyes are studied as fluorescent probes for proteins and nucleic acids detection and visualization owing to their favourable spectral characteristics, namely, the possibility to vary the absorption and emission wavelengths (reaching far-red and even the NIR range), large extinction coefficients, high fluorescence quantum yields, etc. We have shown that far-red bridged pentamethine dyes with cyclohexene groups in the polymethine chain increase their fluorescence intensity dozens of times in the presence of BSA [24]. We have estimated the preferable binding sites of studied dyes on the HSA globule by using molecular docking
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