Molecular characterization of the β-lactoglobulin conjugated with fluorescein isothiocyanate: Binding sites and structure changes as function of pH

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Abstract

Protein conjugated with dyes is a method which can be used for analyzing food components. For example β-lactoglobulin (βlg) can be conjugated with amine-reactive dyes to form βlg-dye conjugates. In this study, the effect of pH on the conjugation of βlg with fluorescein isothiocyanate (FITC) was investigated using MALDI-TOF MS, LC-MS, dynamic light scattering (DLS) and fourier transform infrared spectroscopy (FTIR). The results showed that the binding numbers increased with the increase in pH, which leading to a greater change in the zeta-potential and the secondary structure of βlg after dye conjugation. In particular, the degree of labelling (DOL) was 94.9 ± 7.9%, and the conjugation was mono-labelled at pH 8, indicating no significant changes in the physicochemical properties of βlg. Furthermore, LC-MS revealed that the most probable conjugated lysine is located at position 100, 47 and 77 of βlg.

Introduction

In the past decades, whey protein isolate (WPI) have garnered great interest and developments in the area of food additives, cosmetic and medicine, because of its special foaming property, emulsifying property and water-holding capacity [[1], [2], [3]]. In particular, β-lactoglobulin (βlg) is the major protein in the whey proteins of the milk of ruminants (~60%) [[4], [5], [6], [7]]. βlg and its conjugates are used as ingredients in the treatment of hypertension, anti-inflammatory adjuvant or as a detection biomarker [[8], [9], [10]]. βlg conjugated with fluorescent dyes is an indirect and novel method when it is used as biomarker in the diagnosis of food hypersensitivity. It allows the rapid identification and concentration detection in protein mixtures according to the conjugated dye fluorescence emission intensity, which can be used as a new analytical approach for food additives and food safety [11,12]. Despite the rapid development of protein biomarkers, our knowledge of their potential biological risk in food, and the effect of the conjugation on functional properties of the protein is still not clear; which is a significant hurdle for the application of protein-dye conjugates. Some recent researches reported that protein conjugation influences the protein structures and surface charges, and thus influences their functional properties and bioactivities, especially in the case of biomarkers made of protein conjugated with dyes [[13], [14], [15]].

βlg is a small protein made of 162 amino acids residues with an extinction coefficient of approximately 17,210 M−1 cm−1 at 280 nm [16]. It has 15 lysine residues per molecule and most of them are located at the surface, and can act as modified sites for amine-reactive dyes, such as fluorescein isothiocyanate (FITC), tetraethyl rhodamine isothiocyanate (TRITC) and alexa fluor dyes [15,17]. Among these fluorescein, FITC is yellow-orange in color with an emission maximum at 525 nm and is commonly used for conjugation to proteins via amine group, because of its excellent fluorescence quantum yield and biological stability. Its absorption maximum is at 495 nm with an extinction coefficient of approximately 68,000 M−1 cm−1. When βlg is exposed to FITC its surface will be modified by the adsorption of FITC, leading to the formation of βlg-FTIC conjugates through the thiocarbamide bond formed by the ε-NH2 and isothiocyanate group of FITC [7,18], causing some structural and activity changes of the protein. The conjugation of dyes on the lysine residues of proteins is a complex process and depends on several factors, such as the nature of the protein, the location of lysine, and the reaction environment [19].

Several researchers investigated different aspects related to the interaction of proteins. Zhang et al. (2013) used multispectroscopic methods to study the mechanism and conformation of the food dye amaranth with human serum albumin (HSA) [20]. Liu et al. (2014) compared the difference in binding affinities and structures of different dyes conjugated with bovine serum albumin (BSA) [21]. Khan et al. (2015) studied the HSA conjugated with fluorophores using cyanine NHS ester (Cy3 and Cy5) reactive dyes at pH 8 [22]. Degree of labelling (DOL) is one of the main methods for estimating the extent of conjugation, however its accuracy is limited by the binding number of dyes. A comprehensive and detailed investigation of protein conjugated with dyes should be carried out to understand the binding mechanism and to determine the structure changes, binding numbers (N), and the modified sites on the protein surface. pH is identified as a key factor for governing the conjugation and binding numbers of FTIC on the βlg molecule [22]. It is suggested that the conjugation of FITC to protein is very weak and results in low DOL under neutral or acidic conditions. However, under strong basic condition multiple FITC molecules can be conjugated with βlg, influencing its surface charges and structure [[23], [24], [25]].

In this study, βlg and FITC were chosen as model molecules to address the influence of pH (6 to 10) on their conjugation. UV spectrophotometery was used to calculate the DOL, MALDI-TOF MS and LC-MS were used to identify the binding numbers of FITC and the modified sites (lysine) of βlg. Furthermore, zeta-potential measurement was used to detect the surface charge of the labelled βlg at different pH, and fourier transform infrared spectroscopy (FTIR) was used to monitor the change in the secondary structure of βlg before and after dye conjugation.

Section snippets

Materials

βlg (18.4 KD) from bovine milk and trypsin from bovine pancreas were purchased from Sigma Aldrich (USA). Fluorescein isothiocyanate (FITC) was purchased from Lumiprobe-Life Science (USA). PD-10 desalting column was purchased from GE Healthcare (USA). Other chemical reagents were of analytical grade and were purchased from Sinopharm Chemical Reagents Co., Ltd. (China).

Conjugation of βlg and FITC

βlg conjugation with FITC was carried out by incubating the protein solution (2 ml, 0.1 mM) with the FITC solution (200 μl,

Degree of labeling (DOL)

DOL is a widely used although it is not an accurate method for estimating the conjugation of proteins and ligands as it relies on the use of the Beer-Lamber law in a liquid [26,28]. Fig. 1 shows the Uv-visible spectrum from 265 nm to 565 nm for FITC solution and βlg-FITC conjugates at different pHs. The results show that the βlg-FITC conjugates contains two absorption peaks, one at 280 nm that belongs to βlg, and the other at 495 nm that belongs to FITC, which increase with the increase in pH (

Conclusion

The conjugation of dyes with proteins is a complex process and depends on many factors, such as the nature of the proteins, the location of lysine, and the reaction environment. The pH that maintains the amine group of βlg in a non-protonated form is identified as a key parameter which governs the conjugation and the binding numbers of FTIC onto the βlg molecule. In this work, the binding numbers of FITC on a single βlg molecule increased with the increase in pH. The DOL and binding number was

Declaration of competing interest

The authors declare no competing financial interests.

Acknowledgement

This work was supported by the National Natural Science Foundation of China (no. 31301470) and Key Research and Development Plan of Shandong Province (no. 2018GNC110029).

References (41)

  • I. Kutzli et al.

    Formation of whey protein isolate (WPI)-maltodextrin conjugates in fibers produced by needleless electrospinning

    J. Agr. Food Chem.

    (2018)
  • P.Y. Madhav et al.

    Formation of corn fiber gum-milk protein conjugates and their molecular characterization

    Food Hydrocoll.

    (2012)
  • A. Jabed et al.

    Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk

    P. Natl. Acad. Sci. USA

    (2011)
  • X.Q. Phoebe et al.

    Molecular characterization of interacting complexes and conjugates induced by the dry-state heating of β-lactoglobulin and sugar beet pectin

    Food Hydrocoll.

    (2019)
  • E. Allard et al.

    Core-shell type dually fluorescent polymer nanoparticles for ratiometric pH-sensing

    J. Polym. Sci. Pol. Chem.

    (2010)
  • F. Bamdad et al.

    Anti-inflammatory and ntioxidant properties of peptides released from β-lactoglobulin by high hydrostatic pressure-assisted enzymatic hydrolysis

    Molecules

    (2017)
  • W. Jiang et al.

    Arginase inhibition protects against hypoxia-induced pulmonary arterial hypertension

    Mol. Med. Rep.

    (2015)
  • O. Trenchevska et al.

    Mass spectrometric immunoassay for quantitative determination of protein biomarker isoforms

    J. Proteome Res.

    (2010)
  • K.D. Volkova et al.

    Hydroxy and methoxy dubstituted thiacarbocyanines for fluorescent detection of amyloid formations

    J. of Fluoresc.

    (2011)
  • M. Molinos et al.

    Development of a hybrid dextrin hydrogel encapsulating dextrin nanogel as protein delivery system

    Biomacromolecules

    (2012)
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