Aggregation of trypsin and trypsin inhibitor by Al cation

doi:10.1016/j.jphotobiol.2017.02.018

Journal of Photochemistry and Photobiology B: Biology

Volume 169, April 2017, Pages 7-12

https://doi.org/10.1016/j.jphotobiol.2017.02.018 Get rights and content

Highlights

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The aggregation of trypsin and trypsin inhibitor by Al cations was reported here.
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Al-protein bindings are via hydrophobic, H-bonding and van der Waals.
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More hydrophobic trypsin inhibitor forms more stable aggregate than trypsin.
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Al cations forced trypsin and trypsin inhibitor into large and robust aggregates.
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Al interaction induced larger perturbations of trypsin inhibitor than trypsin

Abstract

Al cation may trigger protein structural changes such as aggregation and fibrillation, causing neurodegenerative diseases. We report the effect of Al cation on the solution structures of trypsin (try) and trypsin inhibitor (tryi), using thermodynamic analysis, UV–Visible, Fourier transform infrared (FTIR) spectroscopic methods and atomic force microscopy (AFM). Thermodynamic parameters showed Al-protein bindings occur via H-bonding and van der Waals contacts for trypsin and trypsin inhibitor. AFM showed that Al cations are able to force trypsin into larger or more robust aggregates than trypsin inhibitor, with trypsin 5 ± 1 SE (n = 52) proteins per aggregate and for trypsin inhibitor 8.3 ± 0.7 SE (n = 118). Thioflavin T test showed no major protein fibrillation in the presence of Al cation. Al complexation induced more alterations of trypsin inhibitor conformation than trypsin.

Graphical abstract

Introduction

Extensive studies have been reported on the effect of aluminum on human health and disease [1]. Al accumulation in tissues and organs results in their dysfunction and toxicity, which can be correlated with the local concentration of the Al cation [2], [3], [4]. Al is known to induce formation of beta-sheet-rich fibrils that disrupt tissue structure and cause disease. Although not fully proven, Al accumulation in the brain is proposed to be associated with neurodegenerative diseases, including Alzheimer's dementia, Parkinson's disease [3]. It has been shown that metal ions including Al cation may trigger protein structural changes such as aggregation and fibrillation [5], [6]. This study was designed to determine the effect of Al cations on the structural transformations of trypsin and trypsin inhibitor.

Trypsin a water soluble globular protein is a proteolytic enzyme that cleaves peptide bonds at the carboxylic groups of arginine and lysine [7]. Trypsin inhibitors are classified as small proteins or polypeptides that exhibit inhibitory activity against trypsin and can lead to certain diseases in animals and humans [8]. The inhibitory role of trypsin inhibitors comes from their bindings to trypsin and other proteins, causing major protein structural changes [9], [10]. The hydrophobic and hydrophilic characteristics of trypsin and trypsin inhibitor are well known and their effects on enzyme-substrate interactions have been investigated [11], [12], [13], [14], [15], [16]. Trypsin inhibitor with a large hydrophobic region showed different affinity than trypsin towards ligand interactions. Reports show hydrophobicity plays a major role in protein-protein and protein-polymer interactions [10], [11], [12], [13], [14], [15], [16], [17].

We report the results of thermodynamic analysis, spectroscopic studies and AFM imaging for Al cation complexation with trypsin and trypsin inhibitor in aqueous solution at physiological conditions. Structural information regarding Al-protein binding and the effect of cation interaction on protein aggregation and fibrillation are presented here.

Section snippets

Materials

Trypsin from bovine pancreas (MW = 23.8 kDa) and trypsin inhibitor type-1S (MW = 24 kDa) from glycine, max soyabean were purchased from Sigma Chemical Company (St-Louis, MO) and used as supplied. Hydrated AlCl₃ was from Aldrich Chemical Co and used as supplied. Other chemicals were of reagent grades.

Preparation of Stock Solutions

Solutions of trypsin (in H₂O) and trypsin inhibitor (in ethanol/H₂O 25/75%) 120 μM were prepared and diluted to various concentrations in 10 mM Tris-HCl (pH 7.4). Hydrated AlCl₃ was dissolved in 10 mM of

AFM Analysis and Protein Aggregation by Al Cation

We observed the presence of aggregates on the mica surface for Al-protein complexes (Fig. 1). A protein concentration of 1.2 μM was used for incubation to limit unwanted crowding of the aggregates on the surface. As a result we were able to measure the average volume of the protein aggregates and estimate the number of protein per aggregates assuming that all the water is removed upon drying. For trypsin we obtained 5 ± 1 SE (n = 52) proteins per aggregate and for trypsin inhibitor 8.3 ± 0.7 SE (n =

Conclusions

Al-protein bindings are mainly via H-bonding and van der Waals contacts for trypsin and trypsin inhibitor. More stable complexes formed with trypsin inhibitor than trypsin. Major protein aggregation was observed in the presence of Al cations. Al-protein interactions induced more alterations of trypsin inhibitor conformation than trypsin, leading to protein aggregation and a partial protein unfolding.

Acknowledgments

This work is supported by grant from Natural Sciences and Engineering Research Council of Canada (NSERC).

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Aggregation of trypsin and trypsin inhibitor by Al cation

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Preparation of Stock Solutions

AFM Analysis and Protein Aggregation by Al Cation

Conclusions

Acknowledgments

Int. J. Pharm.

Inorg. Biochem.

J. Biol. Chem.

J. Mol. Biol.

Ultrason. Sonochem.

J. Mol. Struct. (THEOCHEM)

Colloids Surf. B

Eur. Polym. J.

J. Colloid Interface Sci.

Colloids Surf. B

Adv. Protein Chem.

Biochim. Biophys. Acta

Int. J. Biol. Macromol.

Biophys. J.

Human Health risk assessment for aluminium, aluminium oxide and aluminium, hydroxide

J. Toxicol. Environ. Health B Crit. Rev.

Aluminium and lead: molecular mechanisms of brain toxicity

Arch. Toxicol.

Influence of curcumin on the Al(III)-induced conformation transition of silk fibroin and resulting potential therapy for neurodegenerative diseases

RSC Adv.

Identity of urinary trypsin inhibitor-binding protein to link protein

J. Biol. Chem.

Comparison of polymer induced and solvent induced trypsin denaturation: the role of hydrophobicity

Colloids Surf. B: Biointerfaces