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Vol 1 | Issue 2 | Jul - Sep 2022                                                                                        Indian J Pharm Drug Studies | 56  

Original Article 

Effect of Glucose Concentrations on the HSA-Hydrochlorothiazide Interaction 

– a study using Circular Dichroism and Molecular Docking  

Marilia Amável Gomes Soares1, Otávio Augusto Chaves 2, Antônio Augusto Fidalgo Neto2, Dilson Silva1, 

Dari Cesarin-Sobrinho3, Célia Martins Cortez1  

From,1Postgraduation in Medical Sciences, Rio de Janeiro State University, Brazil, 2Institute of Chemistry, Federal Rural University 

of Rio de Janeiro  

Correspondence to: Celia Martins Cortez, Rio de Janeiro State University, Postgraduation in Medical Sciences, Av. Prof. Manuel de 

Abreu, 444. CEP 20551-170 - Rio de Janeiro, RJ Brazil. Email: ccortezs@ime.uerj.br  

ABSTRACT 

Diabetes Mellitus is a chronic disease characterized by hyperglycemia and is accompanied by disturbances in the metabolism of 

carbohydrates, lipids, and proteins. It requires a lot of attention, as its complications l ead to serious health damage and even death. 

Hydrochlorothiazide (HCTZ) is a diuretic that is well-tolerated in antihypertensive treatments, it reduces blood volume and, therefore, 

decreases peripheral vascular resistance. However, there are some comments in the literature on the possibility that high blood 

concentrations of blood glucose (GLU) induce tolerance and cause adverse metabolic effects, which might generate clinical 

complications. This study aimed to present results by using circular dichroism (f ar and near UV) to study the interaction between 

human serum albumin (HSA) and hydrochlorothiazide in the absence and presence of glucose at 37ºC. Methods- Experimental data 

were correlated for theoretical analysis through molecular docking simulations. Th e ability of hydrochlorothiazide to bind with HSA 

was verified in normoglycemic (containing glucose 80 mg/dl) and hyperglycemic (containing glucose 320 mg/dl) solutions. Far-UV 

analysis showed that HCTZ caused little perturbation on the secondary structure of albumin for the three HSA: HCTZ ratios (1:12, 1:24, 

and 1:48). We observed a strong disturbance in the secondary structure of albumin was for GLU in hyperglycemic concentration, with 

a maximum reduction of 12.66% at 222 nm. An increase in the blood GLU level causes a functional perturbation in the binding of 

HCTZ with HSA. HCTZHSA binding is spontaneous and causes weak perturbation on the secondary and tertiary structure of albumin, 

however, at high GLU concentration, the perturbation increases which makes the albumin structure unstable. 

Key words: Diabetes Mellitus, Glucose, Hydrochlorothiazide, Human Serum Albumin, CD-Spectroscopy, Molecular Docking 

n the study performed by Soares. Et al we saw the 

interaction of HSA with hydrochlorothiazide (HCTZ) in 

both the absence and presence of glucose using UV-Vis 

absorption and Spectro fluorescence spectroscopy at 37 ºC. 

The study also demonstrated competitive binding using warfarin 

and digitoxin, markers for Sudlow I and Sudlow III sites, 

respectively. As this drug was used in the treatment of 

hypertensive diabetic patients, we were interested in verifying 

if blood glucose (GLU) concentration could interfere with this 

drug-protein interaction. For this, HSA solutions containing 

GLU 80 mg/dl and 320 mg/dl were used named normoglycemic 

and hyperglycemic solutions, respectively. Diabetes mellitus 

(DM) is a chronic disease characterized by high blood sugar 

levels, resulting from reduced or non-insulin production and/or 

reduced tissue sensitivity [2]. Hyperglycemia is accompanied 

by disturbances in the metabolism of carbohydrates, lipids, and 

proteins. It is a disease that requires a lot of attention, as its 

complications can cause serious health damage and even death, 

especially among young people [3] [4]. By its diuretic effect, 

HCTZ reduces blood volume and, therefore, decreases 

peripheral vascular resistance, being in general a well-tolerated 

drug in antihypertensive treatments [5] [6] [7]. 

However, there are some comments in the literature on the 

possibility that high blood concentrations of GLU induce 

tolerance and cause adverse metabolic effects, which might 

generate clinical complications [8] [9] [10] [11].  G However, 

some studies disagree with these opinions [12], suggesting long-

term use and high doses (>50 mg/day) as factors that may be 

behind the adverse effects of HCTZ [13] [14]. The comparison 

of our results of the experiments using spectrofluorescence and 

UV-Vis spectroscopy showed that the microenvironment 

disturbance around aromatic amino acid residues of HSA by its 

interaction with HCTZ was considerably greater in 

hyperglycemic solution than in normoglycemic solution [15]. 

The Fluorescence quenching experiments showed that this drug 

binds to albumin forming a complex in the absence that might 

generate a conformational change in albumin and a reduction in 

the number of sites available for drug binding in the IIA 

subdomain.   

I 



Soares et al                                                                              HSA-HCTZ interactions based on glucose concentrations 

Vol 1 | Issue 2 | Jul - Sep 2022                                                                                        Indian J Pharm Drug Studies | 57  

As the biodistribution and effects of drugs depend on their 

ability to bind to plasma proteins, especially albumin, which is 

the main drug transporter, it becomes relevant to enlarge the 

study on disturbances caused by elevated blood glucose in the 

HSA structure and in the HCTZ-HSA interaction. Circular 

dichroism (CD) spectroscopy is a widespread technique for 

checking perturbation on the secondary (far-UV) and tertiary 

(near-UV) structures of macromolecules upon ligand binding 

[15]. It is known that HSA exhibits two negative bands in the 

far-UV: one at 208 nm (π-π* transition) and the other at 222 nm 

(n-π* transition), which are characteristic of the protein's 

structural units [16]. Complementing spectroscopic analysis, 

which allows obtaining experimental data on the binding 

affinity of a ligand with HSA and theoretical analysis using 

molecular docking explains the binding ability at the molecular 

level, which helps in understanding the experimental data [17] 

[18]. Aiming to complement the results obtained in the previous 

work [1], we studied the interaction of HSA-HCTZ in the 

presence and absence of GLU through the circular dichroism 

technique (far and near UV) also at 37 ºC. Current and previous 

data were correlated to theoretical analysis through molecular 

docking simulations, including the evaluation of the main GLU 

conformation (open or cyclic) in the protein binding pocket.  

MATERIAL AND METHODS   

Materials and Equipment - HCTZ (6-chloro-3-3, 4dihydro-

2H-1,2,4-benzothiadiazole-7-sulfonamide1,1dioxide), D-

glucose, HSA, warfarin, digitoxin as well as phosphate buffer 

solution (PBS) were purchased from Sigma-Aldrich Chemical 

Company, St. Louis, USA. There was no further purification. 

Methanol (spectroscopic grade) was obtained from Vetec, 

Química Fina Ltda, Rio de Janeiro, Brazil. A Millipore Milli-Q 

system (Merck KGaA, Darmstadt, Germany) was used to obtain 

purified water.Circular dichroism (CD) spectra were measured 

on a Jasco J-815 spectrophotometer coupled to a Jasco 

PFD425S15F thermostatic cuvette door with 0.1 ºC accuracy, 

with 3 channels, one for steady-state fluorescence, another for 

the CD and the third for the UV-Vis (Jasco Easton, MD, USA) 

[19].  

Circular Dichroism (CD) Measurements - The circular 

dichroism (CD) analysis was performed using 3.0 ml of HSA 

solution (10-6 M, in PBS, pH=7.4) in the absence and presence 

of HCTZ (1:12, 1:24, and 1:48 HSA-HCTZ solution, in PBS). 

The effect of the GLU concentration on HSA-HCTZ CD spectra 

were evaluated in solutions containing 80 mg/dl and 320 mg/dl 

GLU. All CD spectra were recorded to both far-UV (200-250 

nm, for secondary structure) and near-UV (260-320 nm, for 

tertiary structure) [20, 21]. The intensity of the signals in the CD 

spectra was expressed as molar residue ellipticity (MRE, in deg 

cm2 dmol-1), defined according to the following equation [21].  

,   (3)  

Where θ, n, l, and Cp are the observed ellipticity (mdeg), a 

number of amino acid residues (585 to HSA) [22], optical 

cuvette length (1.0 cm), and molar HSA concentration (10-6 M), 

respectively. In order to calculate the quantitative loss of the 

helical structure of the protein due to HCTZ binding, the MRE 

values at 208 and 222 nm were obtained according to the 

equations [23].  

,   (4)  

,   (5)  

Where 𝑀𝑅𝐸208 and 𝑀𝑅𝐸222 are the significant ellipticity molar 

residues (deg.cm2/dmol) at 208 nm and 222 nm, respectively.  

Molecular Docking Simulations - The crystallographic 

structure of HSA was obtained from Protein Data Bank (PDB) 

with access code 1N5U [22l]. The chemical structure of HCTZ 

and GLU (open and cyclic forms) were built and minimized in 

terms of energy using the Functional Density Theory (DFT) by 

Becke-3-Lee Yang Parr (B3LYP) with the standard base set 6-

31G*, available in the Spartan'14 software (Wavefunction, Inc.) [24].   

 

Figure 1 - Far-UV CD spectra for HSA-HCTZ (A) and HSA-glucose (B) at 37 ºC, respectively. Near-UV CD spectra for HSA-

HCTZ (C) and HSA-glucose (D) at 37 ºC, respectively. [HSA]= 1.00 × 10-6 M and [glucose] = 80 and 320 mg/dl  

    

    



Soares et al                                                                              HSA-HCTZ interactions based on glucose concentrations 

Vol 1 | Issue 2 | Jul - Sep 2022                                                                                        Indian J Pharm Drug Studies | 58  

 

Figure 2 - Far-UV CD spectra for HSA-HCTZ in the presence of glucose, 80 mg/dl (A) and 320 mg/dl (B) at 37 ºC. Near-UV CD 

spectra for HSA-HCTZ in the presence of glucose, 80 mg/dl (C) and 320 mg/dl (D) at 37 ºC. [HSA] = 1.00 × 10-6 M and [glucose] 

= 80 and 320 mg/dl.  

Molecular docking studies were performed using GOLD 5.7 

software (Cambridge Crystallographic Data Centre - CCDC) 

[25]. Hydrogen atoms were added to the albumin structure 

according to the data inferred by the program on the ionization 

and tautomeric states. It is known that HSA presents three main 

binding pockets for different ligands (sites I, II, and III, which 

are identified through Trp214, Tyr-411, and Tyr-161 residues 

[23] [26] [27].  In order to identify the main amino acid residues 

involved in the interaction HSA-HCTZ, as well as to evaluate 

the main possible binding site, a spherical 10 Å radius around 

Trp-214, Tyr-411, and Tyr-161 residues was defined, and 

molecular docking simulations were carried out [28].  

Furthermore, the theoretical evaluation of the GLU effect 

(in the open and cyclic forms) on HSA-HCTZ binding was 

simulated, as well as the docking calculations were carried out 

to HCTZ and GLU (in both forms) at the same time under the 

same spherical radius described above. A more positive docking 

score value indicates better interaction, due to the negative sum 

of a series of energy terms involved in the protein binding 

interaction process. The number of genetic operations (crossing, 

migration, and mutation) in each docking run was defined as 

100,000. The scoring function used was “ChemPLP”, which is 

the standard function of the GOLD 5.7 software. The figure of 

the best docking pose for each case was generated with PyMOL 

DeLano Scientific LLC software [29]. 

RESULTS AND DISCUSSION  

Three different cases were analyzed by CD measurements: (I) 

evaluation of the albumin structure upon HCTZ binding, (II) 

evaluation of the albumin structure upon GLU binding, and (III) 

GLU effect on HSA-HCTZ CD spectra.  Figure 1 A shows that 

in the far-UV, HCTZ caused little perturbation on the secondary 

structure of albumin for the three investigated HSA: HCTZ 

ratios (1:12, 1:24, and 1:48). The content of α-helix (%) at 208 

and 222 nm (Table 1) confirms that HCTZ caused little 

perturbation in the secondary structure of albumin, since, for the 

1:48 ratio, the maximum reductions of 3.47% and 3.79% at 208 

and 222 nm, respectively, were found [30].  

In the absence of HCTZ, GLU at normoglycemic 

concentration also caused a weak disturbance, with a maximum 

reduction of 3.45% at 222 nm. But a relatively strong 

disturbance in the secondary structure of albumin was observed 

for GLU in hyperglycemic concentration, with a maximum 

reduction of 12.66% at 222 nm (Figure 1B and Table 2). Thus, 

high concentrations of GLU in diabetic patients can 

significantly disrupt the secondary structure of albumin, 

possibly destabilizing the interaction forces (e.g., hydrogen 

bonding) responsible for protein stability [31].  

    

    



Soares et al                                                                              HSA-HCTZ interactions based on glucose concentrations 

Vol 1 | Issue 2 | Jul - Sep 2022                                                                                        Indian J Pharm Drug Studies | 59  

Table 1 - α-helix % for HSA without and in the presence of HCTZ in PBS solution at 37 ºC 

Ratio HSAHCTZ  HSAHCTZ (208 nm)  HSAHCTZ (222 nm) Reduction % (208 nm)  Reduction % (222 nm)  

1:0 54.50 51.49 - - 

1:12 54.04 50.40 0.84 2.11 

1:24 52.92 50.27 2.90 2.37 

1:48 52.61 49.54 3.47 3.79 
  

In addition, the near-UV CD spectra for HSA- HCTZ reveal 

weak signals in the range 290-305 nm, which associates with the 

amino acid residue Trp-214 (Figure 1C). This indicates that the 

binding of HCTZ to the subdomain IIA, which is located at the 

primary site for this drug in HSA [1], does not significantly 

perturb the protein tertiary structure, as demonstrated by the 

slight increase in CD signals. These results suggest that there is 

an increase in hydrophilicity in the Trp-214 environment, the 

same trend observed in the results of steady-state fluorescence 

quenching [32]. Verifying the effect of GLU in pure HSA 

solution in Figure 1D, we observed that the CD signals in the 

Trp-214 region show a significant decrease, mainly in the 

hyperglycemic condition, indicating an increase in the tertiary 

structure of the protein by an increase in hydrophobicity. This 

may be due to a collapse transition which can directly impact 

the capacity of ligands binding to HSA [33]. These results are 

relevant since they are suggesting that high blood GLU levels 

cause conformational changes in the albumin structure, which 

can decrease the ability to bind not only to HCTZ but also to 

other drugs [34].   

Table 2 - α-helix % for HSA without and in the presence of glucose in PBS solution at 37 ºC.  

[glucose]  HSA-glucose (208 nm)  HSA-glucose (222 nm)  Reduction % (208 nm)  Reduction % (222 nm)  

0 55.22 52.12 - - 

80 mg/dl 52.68 50.32 4.60 3.45 

320 mg/dl 47.26 45.52 14.42 12.66 

Table 3 - α-helix % for HSA-HCTZ in the presence of glucose (80 and 320 mg/dl) in PBS solution at 37 ºC  

  80 mg/dl glucose 320 mg/dl glucose 

Ratio HSA-HCTZ  208 nm  222 nm  Reduction % (222 nm)  208 nm  (222 nm)  Reductio n % (222 nm)  

1:0  60.5 0  58.6 9  -  61.4 6  58.9 1  -  

1:12  58.7 3  56.0 6  4.48  54.0 6  51.5 2  12.54  

1:24  58.9 1  55.6 6  5.16  51.9 0  51.0 7  13.30  

1:48  59.4 0  54.8 8  6.49  53.2 5  50.7 9  13.78  
  

The same trend was also observed in the region of the spectrum 

associated with Phe–255 (at 270 nm) and Tyr–275 (at 282 nm) 

residues. The same kind of analysis was conducted for HSA-

HCTZ in the solutions containing GLU 80 and 320 mg/dl. 

Figures 2A and 2B indicate moderate and strong perturbations 

in the secondary structure of HSA for normoglycemic and 

hyperglycemic conditions, respectively [35]. The α-helix 

content at 222 nm (Table 4) clearly shows a higher tendency of 

perturbation on the secondary structure upon HCTZ addition in 

the presence than in the absence of GLU (for HSA: HCTZ = 1:4, 

maximum reduction of 6.49% and 13.78% at 222 nm, in 80 and 

320 mg/dl, respectively). These results are related to 

spectrofluorimetry results that suggested a conformational 

change in the HSA structure caused by GLU in hyperglycemic 

concentration as responsible for the marked decrease in the 

binding capacity of HCTZ to HAS [1]. 

Table 4 - α-helix % for HSA-HCTZ in the presence of glucose (80 and 320 mg/dl) in PBS solution at 37 ºC.  

  80 mg/dl glucose  320 mg/dl glucose  

Ratio HSA-HCTZ   208 nm   222 nm  Reduction % (222 nm)  208 nm 222 nm Reduction % (222 nm)  

1:0  60.5 0  58.6 9  -  61.4 6  58.9 1  -  

1:12  58.7  3  56.0 6  4.48  54.0 6  51.5 2  12.54  

1:24  58.9 1  55.6 6  5.16  51.9 0  51.0 7  13.30  

1:48  59.4 0  54.8 8  6.49  53.2 5  50.7 9  13.78  

For the near-UV CD spectra of HSA-HCTZ in the presence of 

GLU, unusual behavior can be seen (Figures 2C and 2D). In the 

hyperglycemic condition, the tertiary contacts decreased in Phe 

and Tyr regions, due to the decreased αhelical content in the 

GLU concentration range under study, which may be 

responsible for the structure loss [32]. But an opposite effect is 

found in the Trp-214 region (290-305 nm). Overall, the presence 

of GLU destabilized the protein structure, and this destabilizing 

effect of GLU becomes more evident with the increase in the 

drug concentration. Theoretical Evaluation of the Binding 

Ability To identify the possible main binding sites (sites I, II, or 

III, located in subdomain IIA, IIIA, and IB, respectively) [36], 

as well as the amino acid residues involved in the HSAHCTZ 

interaction, and the impact of GLU addition (in open and cyclic 

forms) in the binding ability, molecular docking calculations 

were performed with GOLD 5.7 software.  



Soares et al                                                                              HSA-HCTZ interactions based on glucose concentrations 

Vol 1 | Issue 2 | Jul - Sep 2022                                                                                        Indian J Pharm Drug Studies | 60  

From literature, it is known that in a plasma medium, 

Dglucose is found essentially as a mixture of two anomers 

(roughly one-third of α-D-glucopyranose and about twothirds as 

β-D glucopyranose), with practically no furanose forms [37] and 

the interconversion between open-chain aldehyde and ring 

forms is fast and affected by the medium conditions, being 

energetically favorable in the cyclic than the open form (e.g. for 

GLU, about 99.98% is cyclic in water) [38]. Thus, all molecular 

docking calculations for GLU were carried out only assuming 

β-D-glucose in both cyclic and open forms. Table 5 shows the 

docking score values (dimensionless) for the best docking pose 

at the three main binding sites of the HSA structure available for 

interaction HSA-HCTZ, HSA-GLU, and HSA-GLU.HCTZ.  

Table 5 - Docking score values (dimensionless) for the 

interaction of HSA with HCTZ and glucose at sites I-III.  

Sample  Site I  Site II  Site III  

HSA-HCTZ  58.3  51.1  40.8  

HSA-Open-GLU  56.6  43.5  43.6  

HSA-Cyclic-GLU  47.6  41.8  39.6  

HSA-Open-GLU.HCTZ  64.6  41.2  50.4  

HSA-Cyclic-GLU.HCTZ  49.9  39.0  41.8  

The site I had the highest docking score value, the results of 

molecular docking suggested the subdomain IIA as the main 

binding site for HSA-HCTZ, HSA-Open-GLU, and HSA-

Cyclic-GLU, confirming results from spectrofluorimetric [1]. 

Although GLU in its open form is in a smaller proportion than 

its cyclic form, the results of molecular docking suggested a 

higher docking score for the open than for the cyclic form (56.6 

and 47.6, respectively), probably due to the greater flexibility of 

the open-GLU to adapt to the protein-binding pocket. In 

addition, if HCTZ and GLU simultaneously bind to the same 

protein pocket, the results of molecular docking also suggested 

site I as the main region for this interaction. Furthermore, 

according to the theoretical analysis, the interaction HSA-

OpenGLU.HCTZ is more favorable than HSA-Open-GLU and 

HSA-HCTZ (docking score values: 64.6, 56.6, and 58.3, 

respectively). On the other hand, cyclic-GLU showed the 

opposite trend, decreasing the binding capacity of HCTZ to the 

same binding pocket. Experimental data also indicated a 

negative effect of GLU on the HSA-HCTZ interaction [1], 

which may probably be associated with the fact that GLU is in 

its pyranose form. According to the molecular docking analysis 

(Figure 3 and Table 3), hydrogen bonding and van der Waals 

interactions are the main intermolecular forces involved in 

HSA-HCTZ, HSA-GLU, and HSA-GLU.HCTZ interactions 

(for both open and cyclic forms of GLU).  

As an example, the hydrogen atom of the -NH3+ group of 

Lys194, Lys-198, and Lys-443 residues is a potential donor for 

hydrogen bonding with the sulfonamide group of HCTZ at 

distances of 2.10, 3.50, and 3.70 Å, respectively. On the other 

hand, the oxygen atom of Gln-220, Glu-449, and Asp450 

residues is a potential acceptor for hydrogen bonding with the 

NH-portion of the HCTZ structure at distances of 2.10, 1.60, and 

1.60 Å, respectively. In addition, the amino acid residue Arg-

221 also interacts with the sulfonamide group of HCTZ via 

hydrogen bonding at a distance of 1.80 Å. Finally, van der Waals 

interactions between HCTZ and amino acid residues Trp-214 

and Val-342 at distances of 3.10 and 2.80 Å, respectively, have 

also been observed.  

 Figure 3 - (A) Best docking poses for the interaction of HSA-HCTZ at sites I, II, and III. The zoom representation shows the 

main amino acid residues involved in the interaction HSA-HCTZ at a site I. Best docking pose for the interaction between HSA-

glucose (B) and HSA-GLU.HCTZ (C), respectively for glucose in the open form at a site I. Best docking pose for the interaction 

between HSA-glucose (D) and HSA-GLU.HCTZ (E), respectively for glucose in the cyclic form at the site I. Amino acid residues, 

HCTZ, and glucose in the open and cyclic forms are in cyan, purple, beige and orange colors, respectively. Hydrogen, oxygen, 

nitrogen, chloro, and sulfur are in white, red, dark blue, green, and yellow, respectively.  



Soares et al                                                                              HSA-HCTZ interactions based on glucose concentrations 

Vol 1 | Issue 2 | Jul - Sep 2022                                                                                        Indian J Pharm Drug Studies | 61  

CONCLUSION  

Circular dichroism confirms the decrease in the binding ability 

of HCTZ to albumin with an increase in GLU concentration 

due to the perturbation of the albumin structure. The binding 

HCTZ-HSA is spontaneous and causes weak perturbation on 

the secondary and tertiary structure of albumin, however, at 

high GLU concentration, it was observed an increase of 

perturbation on these structures increases, conducting the 

albumin structure to an instability condition and difficulty the 

bind of HCTZ to the protein binding pocket. There is just one 

primary binding site for HCTZ in subdomain IIA (the site I), 

and in this binding pocket of HSA, hydrogen bonding and van 

der Waals interactions as the key forces for the association. 

Overall, the experimental and theoretical results suggest that 

the increase in the blood GLU level can cause functional 

perturbation in the ability of HCTZ to bind to albumin. It 

suggests that the adverse effects of HCTZ in hyperglycemic 

patients may be related to increased levels of free drug in the 

bloodstream by the decrease of its binding to albumin. In this 

way, effective treatment for diabetic patients with HCTZ is 

linked to adequate control of the glycemic index.  

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Vol 1 | Issue 2 | Jul - Sep 2022                                                                                        Indian J Pharm Drug Studies | 62  

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How to cite this article: Marilia Amável Gomes Soares, 

Otávio Augusto Chaves, Antônio Augusto Fidalgo Neto, 

Dilson Silva, Dari Cesarin-Sobrinho, Célia Martins Cortez. 
Effect of Glucose Concentrations on the HSA-

Hydrochlorothiazide Interaction – a study using Circular 

Dichroism and Molecular Docking. Indian J Pharm Drug 

Studies. 2022: 1(2) 56-62. 

Funding: None                     Conflict of Interest: None Stated 

 

 

 

 

 

 

 

 

 


