Purification of Extracellular Cysteine Protease Inhibitor - Assignment Example

Purification of Extracellular Cysteine Protease Inhibitor (a) Journal of Bioscience and Bioengineering 101, 2 (2006): 166-171. (b) Purification and Characterization of Extracellular Cysteine Protease Inhibitor, ECPI-2, from Chlorella sp. (c) Masanobu Ishihara. (d) 2. Mason, R.W., Sol-chunch, K., and Abrahamson, M. (1998) Biochem J., 330, 833-836 2. Abstract Summary: Using a methodology of various chromatographies, ECPI-2 was purified. The inhibitor was stable under heat in acidic or neutral conditions and inhibited the proteolytic activity of papain, ficin, or chymopapain; but not other tested enzymes. Using gel filtration, the α-chymotrypsin hydrolysis from ECPI-2 was further separated into six fractions. Results indicate that ECPI-2 has multiple reactive sites for papain. 3. The authors attempted to purify the extracellular cysteine protease inhibitor for a culture filtrate of a unicellular algae, Chlorella sp. First, they cultured a strain of Chlorella sp. 4533, separated the filtrate by centrifuge, and concentrated it through evaporation. After assaying the protease activity, they eluted the inhibitor and obtained two active fractions, one of which was the primary research component, ECPI-2. The active fractions of this were pooled, dialyzed and concentrated, and then the protein concentration and carbohydrate content were determined and measured. 4. Discussion of Figures and Tables. Table 1 is the purification summary for ECPI-2, providing comparison of progressively purified elements in terms of protein concentration, total and specific activity, and percentage yield from crude of each step in the purification process. This was performed to purify the inhibitor and demonstrate the increasing level of activity. The first step used a DEAE-cellulose column of 3.5x60 cm and quadrupled the specific activity. Next, after the active elements were pooled, dialyzed and concentrated, they were applied to a Sephadex S-300 column (2x130 cm) which increased specific activity by a factor of almost 5X. Finally, after another evaporator concentration, the inhibitor was applied to a 1x150 cm column of Butyl Toyopearl 650 M, again doubling the specific activity; from crude to final, activity was increased by over 40X, giving the authors evidence of purity. Figure 1 demonstrates the SDS-PAGE analysis where Panel A demonstrates purity indicators for both protein (Lane 1) and carbohydrate (Lane 2). Panel B uses marker proteins of known weight to range and bracket ECPI-2, thereby yielding the molecular mass of the inhibitor. Table 1 and Figure 1 provide evidentiary purification and specific mass and carbohydrate band, allowing the authors to conclude that the inhibitor is a glycoprotein with 33.6% carbohydrate residues. Figure 2 illustrates the pH and temperature stabilities of ECPI-2. This procedure was performed to determine the relative stability of the inhibitor under various environmental conditions. The inhibitor was heated to various temperatures and at different pH levels and then its activity assayed. As shown in the graph, activity was unaffected up to 60ºC for all three levels of buffers (acetate @ pH 1.5, phosphate @ pH 7.0, and glycine @ pH 11.5). At +60ºC, however, the alkaline environment began to reduce the inhibitor’s activity, taking it to nothing at the boiling point. While the neutral pH began degrading at around 80ºC, it remained above 80% active to 100ºC. The acidic environment was completely unaffected by the heat. The authors conclude that the heat stability of ECPI-2 is similar to that of ECPI, but are only able to speculate that this is due to carbohydrate residues. Figure 3 shows the inhibitory activity of ECPI-2 in terms of enzymatic digestion. This was performed to further refine the characteristics of the purified inhibitor against a variety of proteases in a neutral setting (~pH 8.5). This was accomplished by incubating 68 μg/ml of inhibitor with 33 μg/ml trypsin or α-chymotrypsin at 37ºC for 20 minutes. The graph illustrates protease activity in terms of μg and clearly demonstrates the inhibitory effect on papain, chymopapain, and ficin, respectively. Similarly, it shows ECPI-2 as being ineffectual on the other proteases, with only cathepsin B demonstrating a slight decrease in activity at concentrations above ~26 μg. This leads to the conclusion that the inhibitor is most effective on certain cysteine proteases, and prompts the comparison of ECPI-2’s inhibitory spectrum to that of microbial originating inhibitors such as leupeptin and antipain; suggesting ECPI-2 as a unique cysteine protease inhibitor. Because the inhibitor was most effective with papain, Figure 4 plots papain activity in varying substrate concentrations with or without the inhibitor present. The reason for this is because having focused upon the inhibitor and identified the protease most affected, it is appropriate to alter the focus and isolate that protease in a substrate and compare the activity both with the inhibitor present and without it. Using a Lineweaver-Burk plot, the data show enzyme activity in azoalbumin with no ECPI-2 concentration compared to that of a 21 μg, where a linear relationship is implied. Thus, the authors conclude that ECPI-2 inhibits papain noncompetitively. Table 2 is a direct comparison of ECPI and ECPI-2. While both were the most active fractions of the purified Chlorella sp. inhibitor, they differ in various relevant areas. Of all the considerations shown in this table, note the carbohydrate content and the K(i) value of azoalbumin in nM. The possible impact of carbohydrate content has been suggested already, but the physicochemical property of azoalbumin warrants further investigation. Accordingly, the authors come to the conclusion that they should test the proteolytic activity of papain against azoalbumin with varying concentrations of ECPI-2 to determine the effect. This is demonstrated in Figure 5. Figure 5 establishes the effect of papain activity in the presence of ECPI-2 at various concentrations; thus complex formation can be quantified as a molar ratio. This experiment was performed to determine the specific molecular relationship between the enzyme and the inhibitor. The chart demonstrates the result of mixing a fixed amount of papain with various concentrations of ECPI-2; from a residual activity level of 100% at 0, to a 0% activity level at an inhibitor ratio of 0.5. This leads to the conclusion that two papain molecules are fully inhibited by one ECPI-2 molecule; thus, ECPI-2 forms the 1:2 stoichiometric complex with this enzyme. As previously mentioned, ECPI-2 underwent gel filtration to isolate six fractions. This was done so that the fractions (F1-F6) could each be assayed for inhibitory activity and subjected to an SDS-PAGE. Figure 6 demonstrates the results of the purified inhibitor being incubated with α-chymotrypsin at 37ºC for 20 minutes at a pH of 8.5, applied to a TSK gel 3000SWXL column, and the fractions assayed. Panel A charts the absorbance, retention time, and total inhibitor activity of the six fractions; Panel B shows the six lanes of the corresponding fractions stained for protein, Panel C shows the stain for carbohydrate. The authors conclude that F-1 and F-2 demonstrate an inhibitory activity for papain, and have a certain amount of carbohydrate; and that F-4 and F-5 contain carbohydrate-free proteins which also have inhibitory activities for papain. F-3 and F-6, as seen in Figure 6, show no carbohydrates, little proteins, and little-to-no inhibitor activity over time. These facts contribute to the conclusions regarding ECPI-2’s novel characteristics. Finally, Figure 7 demonstrates the results of an experiment that further analyzes those fractions of ECPI-2 which exhibited an inhibitory effect against papain. The reason for this was that evaluating the N-terminal amino acid sequence would yield a molecular mass determination to verify against other cysteine protease inhibitors as well as set up further research parameters. The methodology was to perform Mono Q column chromatography twice on a purified sample of a carbohydrate-free inhibitor with a molecular mass of 55 kDa from the α-chymotrypsin digest of ECPI-2. As can be seen in the figure, as well as the conclusion of the authors, the N-terminal amino acid sequence was not homologous to inhibitors of the cystatin family or to other cysteine protease inhibitors. 5. Article Summary. The authors engaged in this research because, while protease inhibitors have been isolated from mammals, plants, insects, and microorganisms, the precise role of such inhibitors is not clearly understood; and with no earlier reports regarding cysteine protease inhibitors in algae, as well as their potential for application to transgenic plant insect resistance, it was considered important to use a purified inhibitor to compare characteristics. The general techniques were to purify the inhibitor, check the effect of pH and temperature, measure inhibitory activity against various proteases, evaluate the substrate concentration on papain activity both with and without the inhibitor, subject the inhibitor to enzymatic digestion (including gel filtration of α-chymotrypsin into six fractions for further assaying), and evaluating the N-terminal amino acid sequence for inhibitory effect. The authors concluded that; ECPI-2 was heat stable under neutral or acidic conditions, perhaps due to carbohydrate residues; the inhibitor was effective against cysteine proteases such as papain, chymopapain and ficin, but not stem bromelain and cathepsin B; ECPI-2 exhibited inhibitory effect against papain noncompetitively; that ECPI-2 is likely a novel inhibitor and that its structure is stable against the action of glucosidase. The authors consider that while ECPI-2 is possibly a novel cysteine protease inhibitor, more research is needed. They also leave questions open about the precise number of sites effectively reactive for papain, the full degradation effect of glycosidases, as well as an analysis for carbohydrate composition of ECPI-2. 6. Future experiments. I would follow the research questions posed by further investigating ECPI-2’s specific reactive sites and attempting to determine if similar inhibitors could be characterized from other microorganisms. I would also be interested in discovering if it were possible to obtain a cysteine protease inhibitor from animal or plant materials, as well as if a cystatin with more than two reactive sites could be identified beyond sunflower and kininogen strains. Works Cited Ishihara, Masanobu, Tomoaki Shiroma, Toki Taira, and Shinkichi Tawata. “Purification of Extracellular Cysteine Protease Inhibitor, ECPI-2, from Chlorella sp.” Journal of Bioscience and Bioengineering 101, 2 (2006): 166-171. Read More