Principles of Biophysical and Biochemical Characterization of Root Vegetables’ Bioactive Proteins

Abstract

The characterization of root vegetables’ bioactive proteins biophysically and biochemically becomes imperative as they play an incredibly important role in the discovery, development, and formulation of bioactive proteins as biopharmaceutical products. This is because bioactive proteins differ in terms of size, molecular weights, structures, and physicochemical properties. Biophysical and biochemical characterization employs several techniques ranging from simple to complex procedures to give insight into proteins’ high-order structures, functions, and biochemical activities. Owing to the increasing awareness and acceptance of the use of peptides and proteins of root vegetable origin as treatment agents against some debilitatingly chronic diseases, researchers are now exploring an eco-innovative approach to reduce their loss by getting to structurally and functionally characterizing them. Several biophysical and biochemical tools are employed routinely for protein characterization and some of which are ultraviolet-visual (UV-Vis) spectroscopy, high-performance liquid chromatography (HPLC), circular dichroism (CD), intrinsic tryptophan fluorescence (ITF), differential scanning calorimetry (DSC), thermal shift assay (TSA), among others.

1. Introduction

Proteins or bioactive active substance-made drugs have become an integral class of therapeutics serving as auspicious alternatives to treat many diseases that have till now proven recalcitrant to treatment [1]. The nutritional benefits of root vegetables are no longer in doubt nowadays as they are known to be bioactive proteins making them one of the heartiest and healthiest foods around with therapeutic benefits. Hence, they essentially serve as an alternative protein source rich in phytochemicals such as polyphenols, carotenoids, etc. This among other reasons which are environmental and physiological has made an increasing number of people now include one root vegetable or the other in their staple food [2, 3]. The burden of necessity is been placed on the increasing demand for proteins, especially those from plants and in particular root vegetables. This is owing to the growing world’s population which stands at around 6.5 billion and is expected to double by the year 2063. This increased demand for root vegetable protein is further corroborated statistically as two-thirds of the planet’s dietary protein comes from vegetables [4]. There exist a broad range of biological activities exhibited by bioactive proteins, and these activities are responsible for their application and interest in foods, supplements, and medicine [5].

The tremendous attention bioactive proteins have gained over the years is not unconnected with their disease prevention and treatment ability, which is owing to their multi-target health benefits. To this end, it becomes imperative to know their distinctive functionalities by separating them from their natural matrices and carrying out their characterization. This separation brings about the unfolding of their functional groups, which interact with target tissues [6]. For scientists to have a deeper understanding of such (potential or already established) therapeutic candidates’ biomolecular mechanisms and interactions, biophysical and biochemical characterization of specific quality and functions becomes imperative. This is as characterization provides information in respect of identity (structure), purity, potency, safety, and stability. Also, these characterizations give insight and understanding into some of the compelling parameters essential for the maintenance of protein’s activity as well as the conformation of the higher-order structure (HOS). These HOS include the tertiary structure (3-dimensional structure), secondary structure (protein’s folding), and quaternary structure (sub-unit association) [7]. The increasing significance of biophysical analysis in the characterization of therapeutics such as bioactive proteins stems from the fact that it enhances the investigation and characterization of such biomolecules using physical techniques [8]. A typical characterization technique employed or carried out by researchers looked at the various structural make-up, functionalities, and stability of a bioactive protein. This is because they have varying degrees of implications as they affect the bioactivity of such bioactive proteins.

These physical techniques employed circular dichroism (CD), Fourier-transmission infrared (FTIR), spectroscopy, differential scanning calorimetry (DSC), intrinsic and extrinsic fluorescence, and dynamic light scattering, among others to elucidate their spectroscopic, thermodynamic, and hydrodynamic parameters [9, 10, 11]. The significance of a biochemical assay or characterization stems from the fact that this approach helps detect, quantify, and or study biological molecules such as bioactive protein’s binding or activity [12].

2. Root vegetables bioactive proteins

Food proteins, particularly root vegetable bioactive proteins, generally now have scientific bases to be regarded as having nutritional and physiological functionalities, regulated by some encrypted peptides in their native protein sequence [13]. Carrot (Daucus carota L,), onion (Allium cepa L,), and lettuce (Lactuca sativa), among others, are globally seen as belonging to the most common root vegetables which are known to have specialized. Although these compounds or molecules are secondary metabolites that do not contribute to the root vegetables’ vital process, they are however beneficial to many living organisms on health grounds [2]. These molecules or compounds which herein are referred to as bioactive proteins will at one time or the other need to be purified, compounded, and stored by targeting the biophysical properties of these bioactive proteins [14]. Root vegetables’ bioactive proteins vary in the bioactive compound from one root vegetable to the other with some conferring characteristic color, taste, etc. For example, while carrot’s (D. carota L,) characteristic orange color is largely due to the β-carotene presence in it, onion’s (A. cepa L,) antioxidant, anti-inflammatory, and antimicrobial properties on the other hand have been linked to the presence of biologically active phytochemicals such as flavonoids, etc. [15]. Potato (Solanum tuberosum L,) as a root vegetable is also rich in bioactive phytochemicals such as β-carotene or carotenoids, ascorbic acids, polyphenols, and natural phenols among others, which determine the color of the potato’s skin and pulp [16].

3. Protein characterization: principles and tools

Understanding bioactive proteins’ mechanism of action is an integral component needed for their development as essential pharmaceutical targets and ultimately leading to their use therapeutically by man [17]. In addition to this, and with the advent of several biophysical tools and biochemical characterization techniques, much vital information embedded in root vegetable bioactive proteins can now be explored. This development is essential for both preliminary research and the commencement of the drug discovery process employing root vegetables’ bioactive proteins [17]. Owing to the increasing awareness and acceptance of the use of peptides and bioactive proteins of plant origin such as root vegetables’ bioactive compounds as treatment agents for some debilitatingly chronic diseases, manufacturing sectors are now exploring an eco-innovative approach to reducing the loss of these bioactive proteins (Table 1) [18, 19].

Target	A biophysical or biochemical approach	Typical information that shows the acceptability
1. Identity	Amino acid analysis & sequencing LC-MS (liquid chromatography-mass spectrometry) Peptide mapping to identify post-translational modifications (PTMs) (eg phosphorylation)	Exact, correct sequence identified Correct relative molecular mass (Mr) within instrument error Number & sites of phosphorylation; extent of phosphorylation
2. Purity	SDS-PAGE (sodium dodecyl sulfate-polyacrylamide electrophoresis)/native PAGE Dynamic laser light scattering (DLS) Analytical gel filtration Analytical ultracentrifugation (AUC)	Single band on a gel; still a single band at high loading Monodisperse, Mr. ± 20% expected Defined a single Gaussian peak for a monomer Indicates homogeneity & correct M
3. Concentration	Ultraviolet (UV) spectrum Bradford assay	Peak at 280 nm; Peak at 205 nm; No peaks above ~340 nm; Test for light scattering (look into ratio at different wavelengths eg A280/A230); concentration calculated using ε Linearity with BSA standards
4. Functionality	Functional assay Isothermal calorimetry (ITC) Surface plasmon resonance (SPR) Functional comparison between protein batches Validity of construct	Functional activity observed with expected parameters (eg kcat, Km, kcat/Km) With known tool ligand: n ± 15% of expected; Kd within 2-fold of reference value; ∆H within 1 kcal/Mol Direct binding assay (DBA): Kd within 2-fold of reference value; Expected theoretical Rmax; Inhibition in solution assay (ISA): [Protein] within ±15% of two different concentration measures (Bradford & A280); competition observed between target definition compound (TDC) and TDC in solution Compare Kd, ∆H, stoichiometry, Km, kcat, kcat/Km (usually >10^6 s^−1 M^−1), Ki; Single phase kinetics Compare Kd, Km, Ki, ∆H with full-length protein; compare structure-activity relationship (SAR)
5. Stability	Differential scanning calorimetry (DSC) Differential scanning fluorimetry (DSF) Selwyn’s test	Good pre-transition baseline; visible Tm (above 37°C); good post-transition baseline Good pre-transition baseline; visible Tm (above 37°C); good post-transition baseline Overlay of plots of [P] vs. [E].t for different combinations of [E] and t

Table 1.

Potential biochemical and biophysical approaches for protein quality control checks [20].

Hence, biophysical tools come in handy to provide critical information in respect of the characterization and behavior of these root vegetable proteins. Generally, protein characterization is not only an incredible aspect of the manufacturing of biopharmaceutical products, but it also plays a significant role in the discovery and development of such pharmaceuticals into ready-to-use therapeutics. This is because proteins or bioactive compounds differ in respect of size, molecular structure, and physicochemical properties. Hence, characterizing proteins allows researchers to have information through the protein identification, profiling, and quantification of the protein’s major and minor components. Also, because a typical bioactive protein must possess a unique three-dimensional structure for it to elicit its beneficial biological activities, and due to the possibility of substantial molecular structure changes, characterizing bioactive protein then becomes a necessity [5]. The various biophysical and biochemical characterization techniques available are broadly tabulated as seen in Figure 1 below.

Figure 1.

Checklist of a typical characterization of choice looked into a bioactive protein.

4. Biophysical characterization of root vegetables bioactive proteins

Biophysical techniques though eclectic is a veritable tool that gives insightful information in respect of biological molecules’ electronic structure (size and shape), dynamics, polarity, as well as a mode of interaction [17]. Physical sciences techniques, principles, application, and study of biological systems have progressively been on the front burner of biological research for the determination of protein’s structural and dynamic properties. This has undoubtedly led to expanding the understanding of their nature, mechanism, and functional roles [21]. Biophysical methods of characterization include several techniques that directly measure the structure, properties, dynamics, or function of biomolecules such as those of bioactive proteins from root vegetables [20]. Some available biophysical tools are suitably handy to be employed to assess root vegetable bioactives’ protein information, and data, as well as interpret data regarding their structure, solubility, size, etc. [17, 22]. Ultraviolet-visible (UV-Vis) and fluorescence spectroscopy, dynamic light scattering (DLS), differential scanning calorimetry (DSC), intrinsic tryptophan fluorescence (ITF), thermal shift assay (TSA), and size exclusion chromatography (SEC) are examples of simple biophysical methods [14]. These biophysical characterizations among others look at the bioactive protein’s higher-order structure such as secondary, tertiary, quaternary, and oligomeric structure, stability, and solubility.

Differential scanning calorimetry is perhaps the only technique of characterization that provide complete thermodynamic parameters of a substance such as a bioactive protein. This is as DSC, a technique that measures the thermal molecular stability and structure of a protein by quantifying the enthalpy (∆H), transition temperature (T_m), and changes in heat capacity (∆Cp), which are parameters protein’s primary structure cannot reveal, are thus elucidated employing this technique [23]. Bioactive proteins like typical protein, interacts, lives, function, and die in a highly crowded environment. This protein interacts with other proteins or molecules called binding is essential for its biological functionalities. Hence, isothermal titration calorimetry (ITC) as a biophysical technique measures the energetic changes that occur as a result of binding between two proteins with heat either released or absorbed. This technique, therefore, estimates these binding affinity K_A (which may either be favorable or unfavorable), enthalpy (∆H), entropy (∆S), and stoichiometry [24].

5. Biochemical characterization of root vegetables bioactive proteins

Root vegetables’ bioactive protein biochemical characterization involves the estimation of the bioactive content and molecular weight determination using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) techniques [25, 26], centrifugation, two-dimensional electrophoresis mass spectrometry, and circular dichroism [26, 27, 28, 29]. Biochemical characterization involves a determination of the biochemical properties of a sample or biological molecule such as root vegetable bioactive proteins while investigating their enzymatic activities in terms of activation or inhibition [17, 22, 30]. These biochemical characterization techniques give insight into the biochemical functionalities of macromolecules such as root vegetable bioactive proteins. Root vegetables’ bioactive protein can also be elucidated to give their structural information in terms of their precise molecular mass, and N-terminal sequence, among others [28]. There are a couple of experimental techniques that are utilized for this characterization and they include assays that allow for the detection, isolation, and purification of proteins [27, 31]. Effect of pH and temperature assay alongside enzymatic activity, solubility at physiological pH, etc., are some of the techniques employed in the course of biochemical characterization. Other biochemical characterizations that may also be carried out include carrying out the protein’s enzymatic activities and terms of its activation or inhibition [30]. However, owing to the laborious nature of traditional biochemical characterization and despite the introduction of automation-enhanced next-generation sequencing (NGS) technology, biochemical characterization remains a low throughput technique. This then explains why researchers have switched to an alternative means of elucidating protein’s biochemical component, and this involves the use of a computational approach [29, 32, 33]. All of these characterization techniques and those of biophysical characteristics are indispensable to the development of root vegetables’ bioactive proteins as human therapeutics [22].

6. Conclusion

The imperativeness of characterizing bioactive protein from root vegetables has been scientifically established beyond the acknowledgement of their therapeutic benefit. With the continued advancement in the available biophysical and biochemical techniques, more elucidation and insight into the factors that contribute to or affect their functionalities, structural orientation, and stability will continue to be available for researchers, with the intent they can be further improved on and developed as biopharmaceuticals. The various characterization principles and techniques that researchers have employed so far are with the view of ascertaining the authenticity of the nutritional and physiological content cum characteristics of root vegetables’ bioactive protein, and this cannot be complete without biophysical and biochemical characterization. This is so as protein’s higher order structure which can either be affected by environmental changes in a particular way, or when it interacts with other molecules such as ligands, denaturant, glycosylation, and oxidation states, remains a critical parameter in the development of bioactive protein into therapeutics.

Conflict of interest

The authors declare that they have no financial or relationship conflicting interests.