Reversible changes of canavalin solubility controlled by divalent cation concentration in crude sword bean extract

Figures & data

Fig. 1. Effects of magnesium chloride (MgCl₂) and sodium chloride (NaCl) concentrations on canavalin solubility.

Notes: MgCl₂ (A) and NaCl (B) were added to sword bean extract at concentrations of 0–50 mM. MgCl₂ and NaCl were added to the sword bean extract at a concentration of 5 mM (lane 2), 10 mM (lane 3), 15 mM (lane 4), 20 mM (lane 5), 25 mM (lane 6), 30 mM (lane 7), 35 mM (lane 8), 40 mM (lane 9), 45 mM (lane 10), and 50 mM (lane11). Distilled water was used instead of MgCl₂ as a control (lane 1). The mixture was centrifuged, and the supernatant was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arrows show canavalin bands. The proportion of residual canavalin in the supernatant was estimated from band intensity using ImageJ (C). Open and closed circles indicate MgCl₂ and NaCl, respectively. Data were expressed as mean ± standard deviation of three independent experiments.

Fig. 2. Effects of high sodium chloride (NaCl) concentrations on canavalin solubility.

Notes: NaCl was added to the sword bean extract at a concentration of 0–400 mM. NaCl was added to the sword bean extract at a concentration of 50 mM (lane 2), 100 mM (lane 3), 150 mM (lane 4), 200 mM (lane 5), 250 mM (lane 6), 300 mM (lane 7), 350 mM (lane 8), and 400 mM (lane 9). Distilled water was used instead of MgCl₂ as a control (lane 1). Proteins were analyzed (A) and quantified (B) as described in Fig. 1. Data were expressed as mean ± standard deviation of three independent experiments.

Fig. 3. Analysis of canavalin solubility induced by magnesium chloride (MgCl₂).

Notes: The decreasing (A and B) and increasing (C and D) intensity of canavalin band was investigated as described in Fig. 1. (A) MgCl₂ was added to the sword bean extract at a concentration of 2 mM (lane 2), 4 mM (lane 3), 6 mM (lane 4), 8 mM (lane 5), 10 mM (lane 6), 12 mM (lane 7), 14 mM (lane 8), 16 mM (lane 9), 18 mM (lane 10), and 20 mM (lane11). Distilled water was used instead of MgCl₂ as a control (lane 1). (B) MgCl₂ was added to the sword bean extract at a concentration of 20 mM (lane 2), 25 mM (lane 3), 30 mM (lane 4), 35 mM (lane 5), 40 mM (lane 6), 45 mM (lane 7), 50 mM (lane 8), 55 mM (lane 9), and 60 mM (lane 10). Distilled water was used instead of MgCl₂ as a control (lane 1). Proteins were quantified as described in Fig. 1(B and D). Data were expressed as mean ± standard deviation of three independent experiments.

Table 1. Midpoint concentrations for the insolbulization and solubilization of canavalin.

	Insolubilization		Solubilization
	Cm^a (mM)	R^2^b	Cm^a (mM)	R^2^b
MgCl2	3.9	0.998	40.0	0.992
CaCl2	3.9	0.998	50.0	0.996

^a C_m indicates the midpoint concentration. The values were determined by the curve fitting analysis using the command for the sigmoidal equation of a graphic soft, KaleidaGraph 4.6.

^b R² indicates the coefficient of determination for the curve fitting analysis and was estimated using the software.

Fig. 4. Effects of calcium chloride (CaCl₂) concentration on canavalin solubility.

Notes: CaCl₂ was added to the sword bean extract at a concentration of 0–10 mM (A and C) and 10–140 mM (B and D). Proteins were analyzed (A and B) and quantified (C and D) as described in Fig. 1. (A) CaCl₂ was added to the sword bean extract at a concentration of 1 mM (lane 2), 2 mM (lane 3), 3 mM (lane 4), 4 mM (lane 5), 5 mM (lane 6), 6 mM (lane 7), 7 mM (lane 8), 8 mM (lane 9), 9 mM (lane 10), and 10 mM (lane11). Distilled water was used instead of CaCl₂ as a control (lane 1). (B) CaCl₂ was added to the sword bean extract at a concentration of 10 mM (lane 2), 20 mM (lane 3), 30 mM (lane 4), 40 mM (lane 5), 50 mM (lane 6), 60 mM (lane 7), 70 mM (lane 8), 80 mM (lane 9), 90 mM (lane 10) 100 mM (lane11), 120 mM (lane 12), and 140 mM (lane 13). Distilled water was used instead of CaCl₂ as a control (lane 1). Data were expressed as mean ± standard deviation of three independent experiments.

Fig. 5. Solubility changes to insolubilized canavalin.

Notes: Magnesium chloride (MgCl₂; A) and calcium chloride (CaCl₂; B) were added to the sword bean extract at a concentration of 15 mM and 10 mM, respectively. Distilled water was used instead of salt as a control (lane 1). The mixture was separated into the supernatant (lane 2) and precipitate (lane 3). The precipitate was suspended in the same volume of distilled water, and then separated into the supernatant (lane 4) and precipitate (lane 5). The precipitate was also suspended in 60 mM MgCl₂ or 200 mM CaCl₂. The suspension was separated into the supernatant (lane 6) and precipitate (lane 7). The supernatant was diluted 4-fold or 20-fold, respectively. The diluted solution was separated into the supernatant (lane 8) and precipitate (lane 9). Proteins in the precipitate were dissolved in 8 M urea and subjected to 10% SDS–PAGE.

Fig. 6. Solubility changes to solubilized canavalin.

Notes: Magnesium chloride (MgCl₂; A) and calcium chloride (CaCl₂; B) were added to the sword bean extract at a concentration of 60 and 200 mM, respectively. Distilled water was used instead of salt as a control (lane 1). The mixture was separated into the supernatant (lane 2) and precipitate (lane 3). The supernatant was diluted 4- or 20-fold, respectively. The diluted solution was separated into the supernatant (lane 4) and precipitate (lane 5). Proteins in the precipitate were dissolved in 8 M urea and subjected to 10% SDS–PAGE.

Fig. 7. Schematic representation of canavalin solubility.

Notes: Arrows show changes in the concentration of divalent cations. Cross mark indicates no progress. “Low” indicates 15 mM Mg²⁺ or 10 mM Ca²⁺. “High” indicates 60 mM Mg²⁺ or 200 mM Ca²⁺.