According to the conventional view, noncovalent association of small molecules with human serum albumin (HSA) occurs principally at the so-called Sudlow's sites located in subdomain IIA and IIIA. By employing a circular dichroism (CD) spectroscopic approach, it is shown that biliverdin is the specific CD label of an additional drug binding area in subdomain IB. CD competition experiments disclosed the entrapment of a diverse assortment of acidic, neutral, and basic molecules within subdomain IB including anticancer agents (camptothecin, doxorubicin, daunorubicin, teniposide, suramin, tyrosine kinase inhibitors), anticoagulants (dicoumarol), various steroids (bile acids, carbenoxolone), nonsteroidal antiinflammatory drugs, natural substances (aristolochic acid, glycyrrhetinic acid), and synthetic dyes (methyl orange, azocarmine B). These finding imply that subdomain IB can be considered as the third major drug binding region of HSA featured with promiscuous ligand recognition ability. Additionally, subdomain IB is allosterically coupled with the Sudlow's sites, the ligand binding of which is shown to alter the HSA binding mode and affinity of biliverdin and hemin. Brief case studies are presented to illustrate how the evaluation of spectral changes of tetrapyrrole CD probes gains new insight into the HSA binding properties of endogenous as well as pharmaceutical compounds.

Subdomain IB is the third major drug binding region of human serum albumin: toward the three-sites model.

The binding mechanism of molecular interaction between diosmetin and human serum albumin (HSA) in a pH 7.4 phosphate buffer was studied using atomic force microscopy (AFM) and various spectroscopic techniques including fluorescence, resonance light scattering (RLS), UV–vis absorption, circular dichroism (CD), and Fourier transform infrared (FT–IR) spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by diosmetin was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The RLS spectra and AFM images showed that the dimension of the individual HSA molecules were larger after interaction with diosmetin. The thermodynamic parameters, ΔH° and ΔS° were calculated to be −24.56 kJ mol–1 and 14.67 J mol–1 K–1, respectively, suggesting that the binding of diosmtin to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies and denaturation experiments in the presence of urea indicate...

Probing the binding of the flavonoid diosmetin to human serum albumin by multispectroscopic techniques.

Beta-sitosterol is a naturally occurring phytosterol that is widely used to cure atherosclerosis, diabetes, cancer, and inflammation and is also an antioxidant. Here, we studied the interaction of beta-sitosterol, isolated from the aerial roots of Ficus bengalensis, with human serum albumin (HSA) at physiological pH 7.2 by using fluorescence, circular dichroism (CD), molecular docking, and molecular dynamics simulation methods. The experimental results show that the intrinsic fluorescence of HSA is quenched by addition of beta-sitosterol through a static quenching mechanism. The binding constant of the compound to HSA, calculated from fluorescence data, was found to be K(beta-sitosterol) = 4.6 +/- 0.01 x 10(3) M(-1), which corresponds to -5.0 kcal M(-1) of free energy. Upon binding of beta-sitosterol to HSA, the protein secondary structure was partially unfolded. Specifically, the molecular dynamics study makes an important contribution to understanding the effect of the binding of beta-sitosterol on conformational changes of HSA and the stability of a protein-drug complex system in aqueous solution. Molecular docking studies revealed that the beta-sitosterol can bind in the large hydrophobic cavity of subdomain IIA, mainly by the hydrophobic interaction but also by hydrogen bond interactions between the hydroxyl (OH) group of carbon-3 of beta-sitosterol to Arg(257), Ser(287), and Ala(261) of HSA, with hydrogen bond distances of 1.9, 2.4, and 2.2 A, respectively.

Molecular Dynamics Simulation and Binding Studies of β-Sitosterol with Human Serum Albumin and Its Biological Relevance

N-trans-p-coumaroyltyramine (CT) isolated from Physalis minima is a phenolic substance exhibiting many pharmacological activities like potent inhibition of acetyl cholinesterase, cell proliferation, platelet aggregation, and also antioxidant activity. Here, we have studied the binding of CT with HSA at physiological pH 7.2 by using fluorescence, circular dichroism spectroscopy, mass spectrometry, and molecular docking methods. From the fluorescence emission studies, the number of binding sites and binding constant were calculated to be 2 and (4.5 +/- 0.01) x 10(5) M(-1), respectively. The free energy change was calculated as -7.6 kcal M(-1) at 25 degrees C, which indicates the hydrophobic interactions of CT with HSA and is in well agreement with the computational calculations and molecular docking studies. The changes in the secondary structure of HSA after its complexation with the ligand were studied with CD spectroscopy, which indicated that the protein became partially unfolded. Also, temperature did not affect the HSA-CT complexes. The binding of CT with HSA was detected as 2 molecules bound to HSA was determined using micro TOF-Q mass spectrometry. Further, molecular docking studies revealed that CT was binding at subdomain IIA with hydrophobic interactions and also by hydrogen-bond interactions between the hydroxyl (OH) group of carbon-16 and carbon-2 of CT and Arg222, Ala291, Val293, and Met298 of HSA, with hydrogen-bond distances of 2.488, 2.811, 2.678, and 2.586 A, respectively.

Interaction Studies of Coumaroyltyramine with Human Serum Albumin and Its Biological Importance

Human serum albumin (HSA) is one of the most widely studied proteins and is an important plasma protein responsible for binding and transport of many exogenous and endogenous drugs. Coumarin derivatives play a critical role as anticancer, antidiabetic, anticoagulant, and analgesic agents. Here we have studied the cytotoxic activity of 7-hydroxycoumarin derivatives (7HC-1, 7HC-2, and 7HC-3) on mouse macrophage (RAW 264.7) cell lines. These studies revealed that 7-hydroxycoumarin derivatives caused an increased inhibition in growth of inflamed macrophages in a concentration-dependent manner with an IC50 of 78, 63, and 50 μM. Further studies, using fluorescence, circular dichroism spectroscopy, molecular docking, and molecular dynamics methods, show binding of 7HC (umbelliferone) derivatives with HSA at physiological pH 7.2. The binding constant of 7HC derivatives with HSA obtained from fluorescence emission was found to be K7HC-1 = 4.6 ± 0.01 × 104 M–1, K7HC-2 = 1.3 ± 0.01 × 104 M–1, and K7HC-3 = 7.9 ± 0.01...

Binding and molecular dynamics studies of 7-hydroxycoumarin derivatives with human serum albumin and its pharmacological importance.

Betulinic acid (BA) has anti cancer and anti-HIV activity and has been proved to be therapeutically effective against cancerous and HIV-infected cells. Human serum albumin (HSA) is the predominant protein in the blood. Most drugs that bind to HSA will be transported to other parts of the body. Using micro TOF-Q mass spectrometry, we have shown, for the first time that BA isolated from a plant (Tephrosia calophylla) binds to HSA. The binding constant of BA to HSA was calculated from fluorescence data and found to be KBA = 1.685 ± 0.01 × 106 M−1, indicating a strong binding affinity. The secondary structure of the HSA–BA complex was determined by circular dichroism. The results indicate that the HSA in this complex is partially unfolded. Further, binding of BA at nanomolar concentrations of BA to free HSA was detected using micro TOF-Q mass spectrometry. The study revealed a mass increase from 65199 Da (free HSA) to 65643 Da (HSA + drug), where the additional mass of 444 Da was due to bound BA. Based on the results of this study, it is suggested that micro TOF-Q mass spectrometry is useful technique for drug binding studies.

Betulinic acid binding to human serum albumin: A study of protein conformation and binding affinity

Human serum albumin (HSA) is a predominant protein in the blood. Most drugs can bind to HSA and be transported to target locations of the body. For this study, we have extracted 3- trans -feruloyl maslinic acid (FMA) from the medicinal plant Tetracera asiatica , its a non-fluorescent derivative have potent anti-cancer, anti-HIV, anti-diabetic, and anti-inflammatory activities. The binding constant of the compound with HSA, calculated from fluorescence data, was found as K FMA  = 1.42 ± 0.01 × 10 8  M −1 , which corresponds to 10.9 kcal M −1 of free energy. Furthermore, microTOF-Q mass spectrometry data showed binding of FMA at nanomolar concentrations of FMA to free HSA. The study detected a mass increase from 66,560 Da (free HSA) to 67,919 Da (HSA + drug). This indicated a strong binding of FMA to HSA, resulting in an increase of the protein’s absorbance and fluorescence. The secondary structure of HSA + FMA (0.1 mM) complexes showed the protein secondary structure became partially unfolded upon interaction of FMA with HSA, as well as indicating that HSA–FMA complexes were formed. Docking experiments uncovered the binding mode of FMA in HSA molecule. It was found that FMA binds strongly in different places with hydrogen bonding at IB domain of Arg 114, Leu 115 and Asp 173.

Anilkishor Gollapudi

Papers

Betulinic acid binding to human serum albumin: A study of protein conformation and binding affinity

Novel binding studies of human serum albumin with trans-feruloyl maslinic acid