Molecular and practical aspects of the enzymatic properties of human serum albumin and of albumin–ligand complexes

Kragh‐Hansen, Ulrich

doi:10.1016/j.bbagen.2013.03.015

Cited by 103 publications

(85 citation statements)

References 87 publications

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“…Although serum is a possible source of carboxyesterase, and also contains serum albumin, with the potential to generate nonspecific background signal from calcein over time (Kragh-Hansen, 2013; Redinbo and Potter, 2005), the small percentage of CS present in the wells is apparently not enough to interfere with the CaAM assay during the elapsed time for executing the current protocol (Supplemental Data and Figure S1). However, these esterase activities are an important consideration for assaying cells cultivated with serum concentrations above 1% (see Potential pitfalls and troubleshooting , below).…”

Section: Discussionmentioning

confidence: 99%

Streamlined duplex live-dead microplate assay for cultured cells

Pfeffer

Fliesler

2017

Experimental Eye Research

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A duplex fluorescence assay to assess the viability of cells cultured in multi-well plates is described, which can be carried out in the original culture plate using a plate reader, without exchanges of culture or assay medium, or transfer of cells or cell supernatant. The method uses freshly prepared reagents and does not rely on a proprietary, commercially supplied kit. Following experimental treatment, calcein acetoxymethyl ester (CaAM) is added to each well of cultured cells; after 30 min, the fluorescence intensity (emission λmax ∼ 530 nm) is measured. The signal is due to formation of calcein, which is produced from CaAM by action of esterase activity found in intact live cells. Since live cells may express plasma membrane multidrug transport proteins, especially of the ABC transporter family, the CaAM incubation is carried out in the presence of an inhibitor of this efflux process, thereby improving the dynamic range of the assay. Next, SYTOX® Orange (SO) is added to the culture wells, and, after a 30-min incubation, fluorescence intensity (emission λmax ∼ 590 nm) is measured again. SO is excluded from cells that have an intact plasma membrane, but penetrates dead/dying cells and can diffuse into the nucleus, where it binds to and forms a fluorescent complex with DNA. The CaAM already added to the wells causes no interference with the latter fluorescent signal. At the conclusion of the duplex assay, both live and dead cells remain in the culture wells and can be documented by digital imaging to demonstrate correlation of cellular morphology with the assay output. Two examples of the application of this method are provided, using cytotoxic compounds having different mechanisms of action.

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Section: Discussionmentioning

confidence: 99%

Streamlined duplex live-dead microplate assay for cultured cells

Pfeffer

Fliesler

2017

Experimental Eye Research

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“…29) Ethanol induces conformational changes in the surrounding environment at the binding site of HSA. 15,30) We speculate that the effect of ethanol on the hydrolysis of p-nitrophenyl acetate is induced by the changes of microenvironment and conformation in the esterase-like hydrolysis site.…”

Section: Discussionmentioning

confidence: 99%

The Effect of Ethanol on the Hydrolysis of Ester-Type Drugs by Human Serum Albumin

Tatsumi

Inoue

Hamaguchi

et al. 2018

Biological & Pharmaceutical Bulletin

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Human serum albumin (HSA) has two major ligand-binding sites, sites I and II, and hydrolyzes compounds at both sites. Although the hydrolytic interaction of ester-type drugs with other drugs by HSA has been reported, there are only a few studies concerning the effect of pharmaceutical excipients on the hydrolysis of ester-type drugs by HSA. In the present study, we investigated the effect of ethanol (2 vol%; 345 mM) on the hydrolysis of aspirin, p-nitrophenyl acetate, and olmesartan medoxomil, which are ester-type drugs, with 4 different lots of HSA preparations. The hydrolysis activities of HSA toward aspirin, p-nitrophenyl acetate, and olmesartan medoxomil were measured from the pseudo-first-order degradation rate constant (k obs ) of salicylic acid, p-nitrophenol, and olmesartan, respectively, which are the HSA-hydrolyzed products. Ethanol inhibited hydrolysis of aspirin by HSA containing low levels of fatty acids, but not by fatty acid-free HSA. Ethanol inhibited hydrolysis of p-nitrophenyl acetate by both fatty acid-free HSA and HSA containing low levels of fatty acids. In contrast, the hydrolysis of olmesartan medoxomil by HSA was insignificantly inhibited by ethanol, but inhibited not only by warfarin and indomethacin but also by naproxen, which are site I binding drugs and a site II binding drug, respectively. These results suggest that the inhibitory action of ethanol on the hydrolysis of ester-type drugs by HSA differs between site I binding drugs and site II binding drugs.Key words human serum albumin; ethanol; hydrolysis; olmesartan medoxomil; aspirin; drug interaction Human serum albumin (HSA) is a monomeric, 585-residue protein, and contains three structurally similar α-helical domains (I-III); each domain is divided into subdomains A and B, which contain six and four α-helices, respectively.

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“…The ability to unfold and refold (i.e conformational flexibility) is built into the structure of albumin so that it may alter its shape in response to changes in its environment (Kragh-Hansen et al, 2013). This allows albumin to bind many substances and operate as an excellent molecular chaperone (Anand and Mukherjee, 2013).…”

Section: Albuminmentioning

confidence: 99%

“…Albumin has been studied extensively due to its ability to reversibly bind and transport a vast array of molecules throughout the body, and hence influence multiple biological systems (Peters and Stewart, 2013). Albumin has several physiological functions, driven principally by its colloidal properties (Kragh-Hansen, 2013). Its primary function is to regulate blood volume by maintaining the colloid osmotic pressure of the blood compartment.…”

Section: Albuminmentioning

confidence: 99%

Cryptic epitopes and functional diversity in extracellular proteins

Mortimer

Minchin

2016

The International Journal of Biochemistry & Cell Biology

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The functional diversity of proteins is a major factor determining the complexity of cells and tissues. Both translational and post-translational modifications contribute to this diversity. Recently, protein unfolding and refolding has been recognised as another mechanism for diversity by unmasking buried or cryptic sequences (epitopes) that possess physiological functions. In the current review, we focus on extracellular proteins where folding dynamics can be influenced by mechanical forces, protein-protein interactions and denaturation. Many cryptic epitopes in these proteins are exposed following proteolytic cleavage, but recent data indicate that unfolding/refolding play an important role in regulating the physiological behaviour of extracellular proteins. By understanding how and when hidden sequences are exposed, novel techniques for manipulating the function of these proteins may be uncovered.

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Molecular and practical aspects of the enzymatic properties of human serum albumin and of albumin–ligand complexes

Cited by 103 publications

References 87 publications

Streamlined duplex live-dead microplate assay for cultured cells

Streamlined duplex live-dead microplate assay for cultured cells

The Effect of Ethanol on the Hydrolysis of Ester-Type Drugs by Human Serum Albumin

Cryptic epitopes and functional diversity in extracellular proteins

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