## 4 Discussion  
To separate the gingipains into their constituents, a variety of procedures are employed. This is due to the fact that gingipains are the key invasion factor used by these bacteria to cause periodontitis [31]. Therefore, it is critical to isolate and characterize gingipains that enable the researchers to explore and understand their underlying invasion mechanisms [32; 33]. In a previous study, a variety of chromatographic methods were combined to separate the gingipains into their component parts [22]. Besides chromatography techniques, other methods, including cloning the gingipain encoding gene into a bacterial vector that allows them to express the recombinant protein, are also employed for gingipain purification [24; 34]. However, the methods used to isolate the gingipains in the past are  
Figure 4: Zymography gels showed the transparent bands produced by the proteases found in the crude gingipains sample of _P. gingivalis_ HG66. All Lane 1 was BLUeye pre-stained protein standards. (**a**) Crude gingipains samples without any treatment (2 and 3). (**b**) Crude gingipains sample treated with (2) and without (3) Leuepeptin inhibitor. (**c**) Crude gingipains sample treated with (2) and without (3) Cathepsin B inhibitor II. (**d**) Crude gingipains sample treated with (2) and without (3) TLCK inhibitor. time-consuming and difficult. Consequently, this study demonstrated a quick and an easy solution to solve this issue. In the present investigation, reducing SDS-PAGE, non-reducing gelatin zymogram, and Native-PAGE all showed a significantly different amount of protein bands. The protein samples in SDS-PAGE are first reduced by \(\beta\)-mercaptoethanol ethanol and then further boiled at 100 \({}^{\circ}\)C before electrophoresis, which results in a partial or completely dissociation of non-covalently linked gingipains complexes. Multiple bands were seen to develop on the non-denatured SDS-PAGE in earlier research, which led to the conclusion that RgpA and Kgp are made up of numerous domains that are not covalently connected [21; 35; 36]. Whereas the protein samples that are subjected to gelatin zymogram are not reduced and heated showed a slightly higher molecular weight on the gel, which indicates that gingipains are comprised of catalytic domains bound to several hemagglutinin/adhesin domains, which remain intact as a protein complex without the treatment of a reducing agent and boiling [35]. In the present investigation, the number of protein bands and their molecular weight was identical to those found in [PERSON] et al. (1995) [37]. The dissociation of the gingipain's catalytic domain and hemagglutinin/adhesion domains are thus indicated by the reduced molecular weight and appearance of numerous bands on the SDS-PAGE gel [35]. According to earlier research, gingipains are known to be produced and secreted in various forms depending on the strain of _P. gingivalis_[22; 38]. The arginine-specific gingipain, RgpA is a complex protein with approximately 95 kDa to 110 kDa, which is composed of one catalytic domain (\(\sim\)50 kDa) and several non-covalently bonded adhesin chains (44 kDa, 27 kDa, 17 kDa, and 15 kDa) [39]. RgpA is mostly found in the form of single polypeptide molecule with a molecular weight in the range between 70 kDa to 90 kDa. While RgpB, the other arginine-specific gingipain, can exist either in monomeric form as catalytic domain (\(\sim\)48 kDa) or membrane-associated form as mt-RgpB (\(\sim\)70-90 kDa) [25; 26; 40; 41]. The second major proteinase, Kgp, a lysine-specific gingipain, also consists of one catalytic domain (\(\sim\)60 kDa) and several non-covalently linked hemagglutinin/adhesin domains (44 kDa, 30 kDa, 27 kDa, 17 kDa) [39]. Protein elution from an electrophoresed gel using diffusion methods is a relatively simpler and cost-saving method to extract and purify the target proteins [25; 26; 40; 41].