**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].

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.

### **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


**Table 1.**

*Principles of Biophysical and Biochemical Characterization of Root Vegetables' Bioactive Proteins DOI: http://dx.doi.org/10.5772/intechopen.107986*

#### **Figure 1.**

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

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 (Tm), 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 KA (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 [26–29]. 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].
