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Journal of Chinese Integrative Medicine ›› 2011, Vol. 9 ›› Issue (7): 768-774.doi: 10.3736/jcim20110711

• Original Experimental Research • Previous Articles     Next Articles

Analysis on competitive interaction between arenobufagin and verapamil hydrochloride with serum albumin

Guo-di Lu, Jing Zhou, Hong-yue Ma(),De-kang Wu(), Da-wei Qian, Yu-ping Tang, Ming Hong, Yong-qing Hua, Jing-ao Duan   

  1. Jiangsu Key Laboratory for Traditional Chinese Medicine Formulae Research, Nanjing University of Traditional Chinese Medicine, Nanjing 210046, Jiangsu Province, China
  • Received:2011-03-15 Accepted:2011-04-12 Online:2011-07-20 Published:2011-06-15

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Objective: The present study was performed to investigate competitive interaction between arenobufagin and verapamil hydrochloride with serum albumin.
Methods: Equilibrium dialysis and high-performance liquid chromatography were used to analyze the binding rates of the two medicines to serum protein. The interactions based on bovine serum albumin (BSA) and human serum albumin (HSA) were investigated by using spectrofluorimetry. The interaction mode of arenobufagin and verapamil hydrochloride binding to serum proteins was simulated by molecular docking.
Results: The rate of arenobufagin (0.1 μg/mL) binding to bovine serum was (61.1±0.2)%. Verapamil hydrochloride (0.025 to 0.1 μg/mL) significantly reduced the bovine serum binding rate of arenobufagin, from (60.2±3.7)% to (36.9±3.4)%. However, arenobufagin at the tested doses had no marked effects on the binding rate of verapamil hydrochloride. Furthermore, the verapamil hydrochloride had an active effect on the arenobufagin-induced fluorescence quenching of BSA and HSA. The molecular docking results showed that verapamil hydrochloride and arenobufagin binded to HSA at site Ⅰ . Molecular interaction energy of verapamil hydrochloride binding to site Ⅰ was stronger than that of arenobufagin.
Conclusion: Verapamil hydrochloride reduces the binding of arenobufagin to bovine serum. The mechanism may be a competitive interaction of arenobufagin and verapamil hydrochloride at site Ⅰ on HSA.

Key words: arenobufagin, verapamil hydrochloride, bovine serum, bovine serum albumin, human serum albumin, equilibrium dialysis, fluorescence spectroscopy, high-performance liquid chromatography

Table 1

Precision and recovery of arenobufagin and verapamil hydrochloride in serum (%)"

Concentration
(μg/mL)		 n Extraction
recovery (x±s)
 Inter-day
precision
 Intra-day
precision
 Concentration
(μg/mL)		 n Extraction
recovery (x±s)
 Inter-day
precision
 Intra-day
precision
Are Verapamil
0.1 6 69.3±6.7 3.2 7.1 2 6 76.7±4.1 3.1 7.0
0.4 6 75.8±5.2 2.5 1.6 8 6 75.9±7.3 2.5 9.9
4 6 77.3±4.4 3.1 9.3 50 6 67.0±0.4 2.3 4.7

Table 2

Precision and recovery of arenobufagin and verapamil hydrochloride in buffer (%)"

Concentration of
Are (μg/mL)		 Extraction
recovery (x±s)		 Inter-day
precision		 Intra-day
precision		 Concentration of
verapamil (μg/mL)		 Extraction
recovery (x±s)		 Inter-day
precision		 Intra-day
precision
0.1 67.5±5.5 3.1 7.2 1 62.5±2.3 3.5 6.1
0.4 76.0±5.5 2.1 1.7 2 71.5±4.3 3.2 5.9
1 82.3±7.4 3.3 8.0 4 75.2±0.7 3.1 5.6

Figure 1

Fluorescence spectra of human serum albumin and bovine serum albumin at the same arenobufagin concentration in the different concentrations of verapamil HSA: human serum albumin (1.33 mg/mL); BSA: bovine serum albumin (1.36 mg/mL); a-g: verapamil hydrochloride at doses of 0, 1.57, 1.18, 0.785, 0.39, 0.196, 0.098, 0.049 mg/mL + arenobufagin 13.34 mg/mL; h: only BSA or HSA."

Figure 2

Structure diagrams of arenobufagin and verapamil hydrochloride"

Figure 3

Schematic plot of arenobufagin (green) and verapamil hydrochloride (black) binding to HAS (number in protein database: 2BXD; red)"

Table 3

Docking parameters of arenobufagin and verapamil hydrochloride binding to human serum albumin (PDB：2BXD)"

Molecule Docking score Energy of hydrogen
bond (kJ/mol)		 Number of
hydrogen bonds		 Main interaction
of amino acid residue		 Main interaction
of energy (kJ/mol)
MD score
(kJ/mol)		 Rerank score
(kJ/mol)
Are –113.5 –71.1 –2.5 4 Ala291
Are222
Are257
His242
Ile264
Ile290
Leu238
Leu260
Lys195
Lys199
Ser287
Tyr150		 –19.886 9
–6.782 2
–8.136 23
–8.512 1
–4.248 45
–13.801 9
–7.989 61
–6.413 79
–4.552 06
–10.896 1
–5.100 21
–9.321 62
Verapamil –149.7 –91.7 –1.0 1 Ala291
Are257
Glu153
Glu292
Leu238
His242
Leu260
Lys195
Tyr150
Ile290
Ser192		 –14.124
–11.639 8
–13.321 2
–14.649 3
–8.112 2
–5.386 07
–13.579 1
–8.230 84
–9.844 75
–5.634 46
–5.306 01
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