Mass Spectrometric and Spectrofluorometric Studies of the Interaction of Aristolochic Acids with Proteins.

Weiwei Li,Qin Hu,Wan Chan

doi:10.1038/srep15192

Abstract

Aristolochic acid (AA) is a potent carcinogen and nephrotoxin and is associated with the development of “Chinese herb nephropathy” and Balkan endemic nephropathy. Despite decades of research, the specific mechanism of the observed nephrotoxicity has remained elusive and the potential effects on proteins due to the observed toxicity of AA are not well-understood. To better understand the pharmacotoxicological features of AA, we investigated the non-covalent interactions of AA with proteins. The protein-binding properties of AA with bovine serum albumin (BSA) and lysozyme were characterized using spectrofluorometric and mass spectrometric (MS) techniques. Moreover, the protein-AA complexes were clearly identified by high-resolution MS analyses. To the best of our knowledge, this is the first direct evidence of non-covalently bound protein-AA complexes. An analysis of the spectrofluorometric data by a modified Stern−Volmer plot model also revealed that both aristolochic acid I (AAI) and aristolochic acid II (AAII) were bound to BSA and lysozyme in 1:1 stoichiometries. A significantly stronger protein binding property was observed in AAII than in AAI as evidenced by the spectrofluorometric and MS analyses, which may explain the observed higher mutagenicity of AAII.

Highlights

There is significant evidence in the literature that suggests an association between the continuous intake of Aristolochic acid (AA)-containing herbal remedies with the development of “Chinese herb nephropathy” in Belgian women participating in a slimming regimen[8,9]
Similar to many other drugs[12,19,20,21], AA is able to quench the native fluorescence of human serum albumin (HSA)[17]
A similar, but stronger fluorescence quenching effect was observed for aristolochic acid II (AAII) (Fig. 1B), indicating that AAII had a stronger interaction with lysozyme than aristolochic acid I (AAI)

Summary

Introduction

There is significant evidence in the literature that suggests an association between the continuous intake of AA-containing herbal remedies with the development of “Chinese herb nephropathy” in Belgian women participating in a slimming regimen[8,9]. Emerging evidence suggests that chronic food poisoning by AA-contaminated wheat flour is a leading cause of Balkan endemic nephropathy, a peculiar kidney disease that is prevalent among farmers living along the Danube River[7,10,11]. The interactions between proteins and AA are not well understood and investigations into their relationship are relatively new[17]. Interactions of proteins with AAII have not yet been examined in previous studies[17]. We report the protein binding properties of AA using spectrofluorometric and mass spectrometric (MS) methods. Using high-resolution mass spectrometry (HR-MS) coupled with electrospray ionization (ESI), we present the first direct evidence of non-covalent interactions between AA and proteins.

Methods

Results

Conclusion