Proteolytically-induced changes of secondary structural protein conformation of bovine serum albumin monitored by Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy

https://doi.org/10.1016/j.saa.2016.02.013Get rights and content

Highlights

  • Degradation of BSA by serine proteases was monitored with FTIR and CD spectroscopy.

  • Secondary structure analysis was applied to monitor proteolysis process.

  • α-Helical structure of BSA was converted into unordered structure upon digestion.

  • Spectral changes were less during tryptic hydrolysis than chymotryptic one.

  • Correlation of hydrolysis degree with concentration of carboxylates was revealed.

Abstract

Enzymatically-induced degradation of bovine serum albumin (BSA) by serine proteases (trypsin and α-chymotrypsin) in various concentrations was monitored by means of Fourier transform infrared (FT-IR) and ultraviolet circular dichroism (UV-CD) spectroscopy. In this study, the applicability of both spectroscopies to monitor the proteolysis process in real time has been proven, by tracking the spectral changes together with secondary structure analysis of BSA as proteolysis proceeds. On the basis of the FTIR spectra and the changes in the amide I band region, we suggest the progression of proteolysis process via conversion of α-helices (1654 cm 1) into unordered structures and an increase in the concentration of free carboxylates (absorption of 1593 and 1402 cm 1). For the first time, the correlation between the degree of hydrolysis and the concentration of carboxylic groups measured by FTIR spectroscopy was revealed as well. The far UV-CD spectra together with their secondary structure analysis suggest that the α-helical content decreases concomitant with an increase in the unordered structure. Both spectroscopic techniques also demonstrate that there are similar but less spectral changes of BSA for the trypsin attack than for α-chymotrypsin although the substrate/enzyme ratio is taken the same.

Introduction

Proteolysis is a natural process and induces degradation of the protein polypeptide chain by cleaving of peptide bonds by enzymes. Proteolytic enzymes break down the proteins giving rise to the formation of small fragments of proteins such as smaller polypeptides, short peptides as well as isolated amino acids. To date, the substrate specificity of proteolytic enzymes has been studied comprehensively via hydrolysis of low-weight synthetic substrates with one bond [1]. As proteolysis proceeds, it is still ambiguous to characterize the mechanisms and sequence of steps for a proteolysis model since hydrolysis of large biomolecules with multiple bonds is much more sophisticated than the hydrolysis of small-sized synthetic substrates. During proteolysis, observation of released patterns of the polypeptide fragments and tracking of the conformational and secondary structural (α-helix, β-sheet, random coil) changes of the intact proteins may help to solve this puzzling process. Fragments of polypeptide chain in monomeric form or in the form of aggregates provide only a limited accessibility of peptide bonds for the enzyme (masking effect) [2]. Proteolysis as a process of the degradation of protein globule and polypeptide aggregates leads to the increase of the accessibility of peptide bonds (demasking effect). The increase in enzyme accessibility of peptide bonds during proteolysis was analyzed for β-casein hydrolysis by trypsin and casein by chymotrypsin. This was shown in the framework of two-step model with the consecutive demasking and hydrolysis steps [2], [3], [4]. Thus, in the present study, the enzymatic hydrolysis of bovine serum albumin (BSA) has been carried out by using α-chymotrypsin and trypsin that both differ in their preference to cleave a peptide bond.

BSA is the most abundant protein of plasma in the blood circulation system. Its main biological function is to maintain the pH and to regulate the colloidal osmotic pressure of blood [5]. BSA/HSA are perfect shuttles in the body for effector molecules, hormones, fatty acids, bilirubin, steroids, water, Ca2 +, Na+, K+ as well as drugs by binding them and distributing them evenly in the body. It is a single polypeptide chain (66,430 Da) which consists of 583 amino acid residues [6]. The amino acid sequences of BSA are homologous to that of human serum albumin [6], and their identity is about 76% [5], [7]. In this study, BSA was chosen as a substrate due to its well-known structure.

Trypsin and chymotrypsin are extensively used in biotechnology and food industry. These proteolytic enzymes are the members of the serine protease family found in the digestive system, where they split large protein molecules into small pieces [8], [9]. Trypsin has an active site which is responsible for the binding of positively charged lysine and arginine residues [10]. Like trypsin, chymotrypsin has a catalytic triad within its active site which involves Ser195-His57-Asp102 residues. Both proteolytic enzymes have a similar amino acid sequence (about 40%) and have very similar 3-D structures, although their substrate specificities are different. Chymotrypsin has the preference to cut on the carboxyl end of peptide bonds with aromatic and large hydrophobic residues (tyrosine, tryptophan, phenylalanine, methionine and leucine) which fit into a hydrophobic pocket in the enzyme [10] while trypsin predominantly cleaves polypeptides at the carboxyl side of lysine and arginine [9].

The proteolytic enzymes α-chymotrypsin and trypsin attack the protein and cleave them at specific points in their structures, leading to clipping of the intact protein into smaller pieces. Conventionally, the amount of the amino groups is used to determine the degree of hydrolysis [11]. Due to the fact that one amino group (NH2) and one carboxylic group (COOH) are liberated in the result of the hydrolysis of one peptide bond, the number of carboxylate groups is equal to the number of amino groups. Therefore, concentration of carboxylic groups (∆ A at 1593 cm 1) can also be used for the monitoring of the hydrolysis of peptide bonds. Thereby, FTIR spectroscopy can diligently ‘count’ the cleaved peptide bonds. In our previous study [12], we demonstrated sensing of the proteolysis products at around 1593 cm 1 which is an unequivocal indicator for direct monitoring of the proteolysis process by using FTIR spectroscopy. This IR signal absorbing at around 1593 cm 1 corresponds to the free COO groups of cleaved peptide groups.

Fourier transform infrared (FT-IR) and ultraviolet circular dichroism (UV-CD) spectroscopy are applied intensively to determine the secondary structure and conformational changes of proteins. FTIR spectroscopy in the mid-infrared region is an efficient and inexpensive technique for proteolytic studies of proteins and characterization of physicochemical properties of protein hydrolytes as well as susceptibility to proteolysis [12], [13], [14], [15], [16]. The CD spectrum of a protein in the far UV is one of the most commonly used techniques to determine the protein secondary structure content [17]. In this respect, various algorithms have been developed and improved in order to analyze protein secondary structures [18], [19], [20]. In our previous study, hydrolysis of milk proteins was followed for the first time using the FT-IR and UV-CD spectroscopy [12]. Here, we successfully monitored the degradation of a protein in the course of tryptic digestion and the proteolysis products as the resultant of this reaction. This approves that FTIR enables to detect enzymatically-induced small structural changes of proteins as well as proteolysis products as resultant of the hydrolysis of the intact protein over time. The amide I (1600–1700 cm 1) and amide II (1500–1600 cm 1) spectral bands reflect the most pronounced vibrational modes of a protein. These spectral ranges in the mid-IR correspond to the protein secondary structures (α-helix, β-sheet, β-turns, and unordered/random coils) as well as dynamic conformation of the proteins [21], [22], [23]. The H–O–H bending vibration of H2O overlaps with the amide I mode of the protein [23] while the N–H bending vibrations strongly absorb in the spectral region 1620–1580 cm 1 [24]. Importantly, the latter overlaps with the carboxylate group signals absorbing at around 1593 cm 1, an IR signal used to track the proteolysis reaction. In the current study, we used D2O-buffer because of the fact that the amide I band which is mainly composed of the CO stretching mode of polypeptide backbone and the amide II band which arises from coupling of C–N stretching and N–H bending vibrations shift towards lower wavenumbers by 2–10 cm 1 and by ~ 100 cm 1, resp., upon H/D exchange [21], [25], [26].

In the present study, we have utilized FT-IR and CD spectroscopy to figure out a proteolysis model further by tracking the proteolytically-induced conformational changes of an intact protein and the time course of secondary structure alterations as proteolysis proceeds. Here, we have also demonstrated for the first time the correlation between the degree of hydrolysis and the concentration of carboxylic groups measured by FTIR spectroscopy.

Section snippets

Sample preparation

Albumin from bovine serum (Sigma-Aldrich, A7906), trypsin (Sigma-Aldrich, T1426) and α-chymotrypsin (Fluka, 27270) from bovine pancreas of analytical grade were purchased and were used without further treatment. Solutions of all proteins were prepared by dissolving the lyophilized powder in 10 mM potassium phosphate buffer in deuterium oxide (99.9%), and were adjusted to pD 8.4 with DCl and NaOD (pD = 0.4 + pH) [27], [28]. Deuterated protein samples were obtained by equilibrating the protein for 3 h

FTIR spectrum of native BSA

Fig. 1A and D presents the IR absorbance and second derivative spectra of native BSA, respectively, in D2O buffer recorded for 5 h at 37 °C. Native BSA has an amide I band maximum around 1653 cm 1 ascribed to the typical α-helix conformation, as it has been extensively studied with FTIR. BSA is a remarkably stable protein during long-term stability measurements for 5 h at 37 °C. However, the only noticeable alteration over time is that the intensity centred around 1547 cm 1 within the amide II band

Conclusion

The conformational changes induced by proteases occurring in the protein structure were monitored by FT-IR and CD spectroscopy. The overall aim of this study was to demonstrate the applicability of both spectroscopies for the monitoring of the proteolysis process in real time, by following the spectral changes together with secondary structure analysis. In conclusion, both FT-IR and CD spectroscopy clearly reveal that BSA loses its secondary structure elements upon conversion to the peptide

Acknowledgements

Financial support for Mikhail M. Vorob'ev from the Deutscher Akademischer Austauschdienst (DAAD 91578751) is gratefully acknowledged.

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