Molecular interaction of fluorescent carbon dots from mature vinegar with human hemoglobin: Insights from spectroscopy, thermodynamics and AFM

Highlights

• Vinegar carbon dots (VCDs) can bind with human hemoglobin (HHb).

• HHb aggregated after binding with VCDs with slight conformational change.

• The esterase activity of HHb decreased in the presence of VCDs.

Abstract

Foodborne nanoparticles have attracted considerable interest due to their distinctive fluorescence and physicochemical properties. The discovery of vinegar carbon dots (VCDs) has drawn our attention to study their effect on human plasma protein. Herein, spectral, constructional, morphological, and enzymatic activity assessments were carried out to investigate the interaction of VCDs with human hemoglobin (HHb). The intrinsic fluorescence of HHb was quenched significantly by the VCDs through a static quenching process. Furthermore, binding constants and important thermodynamic parameters were calculated, the negative enthalpy and entropy changes were accompanied by a negative Gibbs energy, which proposed the binding between VCDs with HHb was spontaneous. Moreover, negative enthalpy and entropy change corroborated the involvement of van der Waals force and hydrogen bonds in the binding process. Results from FTIR, atomic force microscopy and circular dichroism revealed change of HHB after binding with VCDs although their essential morphological features were unaffected. The esterase activity of HHb decreased after VCDs treatment in a dose-dependent manner, which further confirmed the effect of VCDs on HHb. The results offered detailed information about the interaction between VCDs and HHb.

Introduction

The safety issue of nanomaterials in foods is a long-lasting and hot topic [1,2]. Fluorescent carbon dots (CDs), a new kind of nanoparticles, have been discovered in various foods, including carbohydrate-based food caramels [3], commercial beverages [4,5], coffee [6], grilled hamburgers [7], milk and baked lambs [8,9]. The size of CDs in food items was usually less than 10 nm and bright fluorescence was observed under the irradiation of ultraviolet light [10]. Primary results suggested that the foodborne CDs may cause uncertainty or cytotoxicity on living cells [11,12]. For instance, the CDs formed during the Maillard reaction could enter HepG2 cells and lead to cell death via interfering glycolytic pathway [13]. However, the understanding about the health risk of these foodborne CDs was still limited.

Nanomaterials affect the protein structures or functions through physical binding under the physiological environment, which plays a critical role in nanotoxicity [14]. For example, metal-organic framework nanosheets (MOF) inhibited α-chymotrypsin activity effectively, and the kinetic studies suggested the interaction between MOF and α-chymotrypsin governed the inhibition process [15]. The CDs could also alter the function of protein since the lipase activity was inhibited after CDs addition [16]. Importantly, the interaction between proteins and CDs in food items was reported and the major forces involved in the interaction were proved to be hydrogen bonding and van der Waals forces [11]. Moreover, the CDs isolated from Coca-cola and Pepsi-cola were easily distributed in the digestive tract and were able to cross the blood-brain barrier and enter mouse brain [9]. This result suggests that CDs in food items can be transferred through blood circulation. Therefore, it is important to study the interaction between the protein in the blood and foodborne CDs.

Human hemoglobin (HHb) is the main component of red blood cells, which is composed of globin and heme [17,18]. Besides carrying oxygen and carbon dioxide, HHb could bind with many kinds of substances including exogenous and endogenous ones reversibly. It has been proved that certain exogenous drugs can interact with HHb [19]. Moreover, the interaction between HHb and nanomaterial has been demonstrated as well [[20], [21], [22]]. However, these studies were mainly focused on engineered nanoparticles, the interaction between foodborne nanostructures and HHb, was investigated rarely. Therefore, it is imperative to understand the binding of foodborne CDs with HHb and elucidate their interaction mechanism. The presence of Chinese vinegar carbon dots (VCDs) and their interaction with dopamine were demonstrated in our previous work [23]. However, as one of the most beloved traditional condiments, knowledge is still limited about the interaction between VCDs and HHb. Hence, there is an urgent need to investigate the biological effect of VCDs in blood.

The present work investigated the interaction between VCDs and HHb under physiological conditions. The binding process of HHb and VCDs was analyzed by various techniques to reveal the quenching mechanism of HHb, the number of binding sites and important thermodynamic parameters. The effects of VCDs on structure and function of HHb were examined, and the esterase activity of HHb was evaluated by the p-nitro-phenyl acetate method. The results shed light on the binding mechanism between HHb and VCDs, which provided fundamental information for evaluating the potential effect of nanoparticles from food items.