Rate and Equilibrium Constants for Bupivacaine’s Binding to Isolated Alpha-1-Acid Glycoprotein: An In vitro study.

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Jingzhong Zhang Douglas E. Raines Lawrence P. Cogswell III Gary Strichartz


Binding of local anesthetics to plasma proteins has been presented as an important determinant of their bioavailability. Local anesthetics with a high potential for systemic toxicity, e.g. bupivacaine (BUP), are bound strongly by alpha1-acid glycoprotein (AAG), more weakly by serum albumin, but drug dissociation may be rapid, thus limiting the importance of protein binding. The purpose of this study was to determine the binding kinetics of BUP to AAG. Bupivacaine binding to AAG was monitored by its displacement of the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS). The increased fluorescence of ANS (λ excit/em = 380/480 nm) upon binding to AAG was used to determine the equilibrium and kinetic characteristics of this reaction. By studying how BUP altered the binding kinetics of ANS to AAG it was possible to calculate the BUPs equilibrium and kinetic rate constants for AAG binding. ANS fluorescence increased ca. 50-fold when bound to AAG.  Increasing [BUP] with a constant [AAG] + [ANS] returned ANS fluorescence to its unbound status, due to complete displacement of ANS from AAG; bupivacaine’s competitive equilibrium constant, Ki , equals 1-2 μM (pH 7.4, 23oC). Pre-equilibrating AAG with BUP before the rapid (0.008s) addition of excess ANS slowed the binding of ANS to a rate limited by BUP’s dissociation: koff = 12.0 ± 0.5 s-1, corresponding to a half-time ~0.06 seconds. Therefore, although much of the total serum BUP at toxic levels (2-4 µg/mL) will be bound by plasma proteins, dissociation from the tightest binding protein shows that drug is rapidly freed during organ perfusion, allowing newly unbound drug to permeate into the perfused tissues. The very rapid dissociation of BUP from AAG means that equilibrium binding is a very poor index of bio-availability and systemic toxicity of that local anesthetic.

Keywords: Local anesthetic, bupivacaine, alpha-1-acid glycoprotein, plasma proteins, systemic toxicity

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How to Cite
ZHANG, Jingzhong et al. Rate and Equilibrium Constants for Bupivacaine’s Binding to Isolated Alpha-1-Acid Glycoprotein: An In vitro study.. Medical Research Archives, [S.l.], v. 10, n. 1, jan. 2022. ISSN 2375-1924. Available at: <https://esmed.org/MRA/mra/article/view/2655>. Date accessed: 24 may 2024. doi: https://doi.org/10.18103/mra.v10i1.2655.
Research Articles


1. Wood M and Wood AJ: Changes in plasma drug binding and alpha 1-acid glycoprotein in mother and newborn infant. Clin Pharmacol Ther 1981; 29:522-526
2. Routledge PA: The plasma protein binding of basic drugs.” Br J Clin Pharmacol 1986; 22:499-506
3. Kremer JM, Wilting J and Janssen LH: Drug binding to human alpa-1-acid glycoprotein in health and disease. Pharmacol Rev 1988; 40:1-47
4. Kytta J, Heavner JE, Badgwell JM and Rosenberg PH: Cardiovascular and central nervous system effects of co-administered lidocaine and bupivacaine in piglets. Reg Anesth 1991; 16:89-94
5. Mazoit JX, Orhant EE, Boico O, Kantelip JP, Samii K: Myocardial uptake of bupivacaine: I. Pharmacokinetics and pharmacodynamics of lidocaine and bupivacaine in the isolated perfused rabbit heart. Anesth Analg 1993; 77:469-76
6. Chaplan SR, Bach FW, Shafer SL, Yaksh : Prolonged alleviation of tactile allodynia by intravenous lidocaine in neuropathic rats. Anesthesiology 1995; 83:775-85
7. Dauphin A, Gupta RN, Young YEM and Morton WD: Serum bupivacaine concentrations during continuous extrapleural infusion. Can J Anaesth 1997; 44:367-370
8. Denson D, Coyle D, Thompson G and Myers J. Alpha 1-acid glycoprotein and albumin in human serum bupivacaine binding. Clin Pharmacol Ther 1984; 35:409-415
9. Mazoit JX, Cao LS, Samii K: Binding of bupivacaine to human serum proteins, isolated albumin and isolated alpha-1-acid glycoprotein. Differences between the two enantiomers are partly due to cooperativity. J Pharm Exp Ther 1996; 276:109-115
10. Covino BG, Wildsmith JAW. Clinical pharmacology of local anesthetic agents, Neural Blockade in Clinical Anesthesia and Management of Pain, 3rd edition. Edited by Cousins MJ, Bridenbaugh PO. Philadelphia, Lippincott-Raven, 1998, pp.105-115
11. Parikh HH, McElwain K, Balasubramanian V, Leung W., Wong D, Morris ME and Ramanathan M. A rapid spectrofluorimetric technique for determining drug-serum protein binding suitable for high-throughput screening. Pharm Res 2000; 17:632-637
12. Cogswell LP, Raines DE, Parekh S, Jonas O, Maggio JE, Strichartz GR. Development of a novel probe for measuring drug binding to the F1*S variant of human alpha 1-acid glycoprotein. J Pharm Sci 2001; 90:1407-1423
13. Taheri S, Cogswell LP, Gent A and Strichartz GR: Hydrophobic and ionic factors in the binding of local anesthetics to the major variant of human alpha 1-acid glycoprotein. J Pharmacol Exp Ther 2003; 304:71-80
14. Zini R, Copigneaux C and Tillement JP: 1-anilino-naphthalene 8-sulfonic acid (ANS) as a probe for the binding of antidepressant drugs to human alpha-1-acid glycoprotein (AAG). Prog Clin Biol Res 1989; 300:417-421
15. Johansen AK, Willassen NP and Sager G. Fluorescence studies of beta-adrenergic ligand binding to alpha 1-acid glycoprotein with 1-anillio-8-naphthalene sulfonate, isoprenaline, adrenaline and propranolol. Biochem Pharmacol 1992; 43:725-729
16. Good NE, Winget GD, Winter W, Connolly TN, Izawa S and Singh RM: Hydrogen ion buffers for biological research. Biochemistry 1966; 5:467-477
17. Patel RC, Lange D, McConathy WJ, Patel YC and Patel SC: Probing the structure of the ligand binding cavity of lipocalins by fluorescence spectroscopy. Protein Eng 1997; 10:621-625
18. Lackowicz JR: Fluorescence spectroscopy of biomolecules, Encyclopedia of Molecular Biology and Molecular Medicine. Edited by Meyers A. New York, VCH Publishers, 1995, pp294-306
19. Stryer L: Fluorescence energy transfer as a spectroscopic ruler. Ann Rev Biochem 2978; 47:819-846
20. Herve F, Duche JC, d’Athis P, Marche C, Barre J and Tillement JP: Binding of disopyramide, methadone, dipyridamole, chlorpromazine, lignocaine and progesterone to the two main genetic variants of human alpha 1-acid glycoprotein: evidence for drug binding differences between the variants and for the presence of two separate drug-binding sites on alpha 1-acid glycoprotein. Pharmacogenetics 1996; 6:403-415
21. Solt K, Johansson JS and Raines DE: “Kinetics of anesthetic-induced conformational transitions in a four-alpha-helix bundle protein.” Biochemistry 2006; 45:1435-1441
22. Barbier P, Peyrot V, Dumortier C, D’Hoore A, Rener GA and Engelboroghs Y: Kinetics of association and dissociation of two enantiomers, NSC 613863 (4)- (+) and NSC 618862 (S)-(-) CL980, to tubulin. Biochemistry 1996; 35:2008-2015
23. Flower DR: The lipocalin protein family: structure and function. Biochem J 1996; 318:1-14
24. Schönfeld DL, Ravelli R, Mueller U, Skerra A. The 1.8-A crystal structure of alpha1-acid glycoprotein (Orosomucoid) solved by UV RIP reveals the broad drug-binding activity of this human plasma lipocalin. J Mol Biol. 2008;384(2):393-405. doi: 10.1016/j.jmb.2008.09.020. Epub 2008 Sep 16.
25. Herve F, d’Athis P, Tremblay D, Tillement JP and Barre J. Glycosylation study of the major genetic variants of human alpha 1-acid glycoprotein and of their pharmacokinetics in the rat. J Chromatography B Analyt Technol Biomed Life Sci 2003; 798:283-294
26. Pidikiti R, Zhang T, Mallela KM, Shamim M, Reddy KS and Johansson JS: Sevoflurane-induced structural change in a four-alpha-helix bundle protein. Biochemistry 2005; 44:12128-12135
27. Peters T. All About Albumin: Biochemistry, Genetics and Medical Applications. San Diego, CA: Academic Press Limited. 1996.