Purification of a protein from coelomic fluid of the earthworm Eisenia foetida and evaluation of its hemolytic, antibacterial, and antitumor activities

Figures & data

Figure 1.  The crude coelomic fluid of earthworm was characterized with SDS-PAGE. Lane M: protein ladder; lane A: the filtrated earthworm coelomic fluid (not ultrafiltration); lane B: the ultrafiltrated earthworm coelomic fluid (< 1000 KDa); lane C: the ultrafiltrated earthworm coelomic fluid (5 KDa1–000 KDa); lane D: the ultrafiltrated earthworm coelomic fluid (< 5 KDa).

Figure 2.  Two main components (peak 1 and 2) were obtained after the gel chromatography of the ultrafiltrated earthworm coelomic fluid (5 KDa–1000 KDa) (left). The bioactive protein (peak 1) was collected after the ion exchange chromatography (right).

Figure 3.  Standard curve related to the relative mobility and logarithm of standard molecular weight was established. The x-axis represents the relative mobility, and the y-axis represents the logarithm of the standard molecular weight of protein (protein ladder).

Figure 4.  The purified earthworm proteins were characterized with SDS-PAGE. Lane M: protein ladder; lane A: the ultrafiltrated earthworm coelomic fluid (5 KDa–1000 KDa); lane B: peak 1 purified after the gel chromatography; C: peak 1 (ECFP with molecular weight of 38.6 KDa) purified after the ion exchange chromatography.

Table 1.  Hemolytic activity of the ECFP on CRBC.

Groups	ECFP concentration (µg/mL)	Hemolytic activity
Control	0	−
1	0.19	−
2	0.39	+
3	0.78	+
4	1.56	++
5	3.12	++
6	6.25	++
7	12.5	+++
8	25	+++
9	50	+++
10	100	++++

The CRBC were incubated with ECFP for 2 h, and the results indicated that 0.39 μg/mL was the minimal hemolytic concentration. The symbols “−/+” indicate hemolytic intensity: “−” 0% hemolysis; “+” 25% hemolysis; “++” 50% hemolysis; “+++” 75% hemolysis; “++++” 100% hemolysis.

Table 2.  Antibacterial activity of the ECFP on bacteria.

Groups	ECFP concentration (μg/mL)	Turbidity of colorimetric assay		Oxford cup plate assay
		Escherichia coli	Staphylococcus aureus	Escherichia coli	Staphylococcus aureus
Control	Control	−	−	−	−
1	22.5	−	−	−	−
2	45	−	+	−	−
3	90	+	+	−	+
4	180	+	++	+	+
5	360	++	++	++	++

After bacteria were treated in the presence of ECFP, minimal inhibitory concentrations of 90 μg/mL on Escherichia coli and 45 μg/mL on Staphylococcus aureus were determined, respectively. Furthermore, minimal bactericidal concentrations of 180 μg/mL on Escherichia coli and 90 μg/mL on Staphylococcus aureus were detected. Symbol “−/+” indicated intensity of activity, “−” no activity; “+” activity on bacterial growth than control; “++” remarkable activity.

Figure 5.  HeLa cells and LTEP-A2 cells were exposed to ECFP with final concentration of 10, 20, 40, 80, and 160 μg/mL for 96 h, and MTT method was performed for determining the cell proliferation. Every dosage was repeated three times. The ECFP (38.6 KDa) inhibited the proliferation of HeLa cells (left, IC₅₀ 77 μg/mL) and LTEP-A2 cells (right, IC₅₀ 126 μg/mL) in vitro compared with the control both in a time- and dose-dependent manner.