Preparation of an antitumor and antivirus agent: chemical modification of α-MMC and MAP30 from Momordica Charantia L. with covalent conjugation of polyethyelene glycol

Figures & data

Table 1 Summarization of purification of alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) from 200 g of bitter melon seeds

Steps	Total volume (mL)		Protein content (mg/mL)		Total protein (mg)		Field (%)
Extract-free cells	800		15.0		12,000		100
AS precipitation	100		37.5		3750		32.0
SP Sepharose FF	360		2.3		828		6.9
Sephacryl S-100	110		4.5		495		4.1
Macro-Cap-SP	α-MMC	MAP30	α-MMC	MAP30	α-MMC	MAP30	α-MMC	MAP30
	40	35	5.3	3.5	200	120	1.7	1.0

Notes: Values reported are an average value of five preparations. The data from both crude extraction and precipitation with ammonium sulfate were obtained by the method of Lowry et al;¹⁴ other data were assayed at 280 nm.

Figure 1 SDS-PAGE of extracts and eluates from different purification steps. Lane 1: crude extract; Lane 2: dialytic sample after AS precipitation; Lane 3: eluate from SP-Sepharose FF chromatography; Lane 4: eluate from gel filtration chromatography; Lane 5: α-MMC from Macro-Cap-SP chromatography; Lane 6: MAP30 from Macro-Cap-SP chromatography; Lane7: LMW calibration kit.

Figure 2 Western blot analysis of α-MMC and MAP30. (A) Lane 1: reduced SDS-PAGE of α-MMC; Lane 2: western blot profile of α-MMC. (B) Lane 1: reduced SDS-PAGE of MAP30; Lane 2: western blot profile of MAP30.

Figure 3 MALDI-TOF-MS analyses of both α-MMC and MAP30. (A) MALDI-TOF-MS profile of α-MMC. The average MW of α-MMC was 28551.6 Da (B) MALDI-TOF-MS profile of MAP30. The average MW of MAP30 was 29072.0 Da.

Figure 4 Gradient SDS-PAGE of both PEGylated α-MMC and PEGylated MAP30 conjugates. (A) Lane 1: α-MMC as a control; Lane 2: mixture of α-MMC and α-MMC-PEG conjugate; Lane 3: purified α-MMC-PEG conjugate; Lane 4: unmodified α-MMC; Lane 5: HMW calibration kit. (B) Lane 1: MAP30 as a control; Lane 2: mixture of MAP30 and MAP30-PEG conjugate; Lane 3: purified MAP30-PEG conjugate; Lane 4: unmodified MAP30; Lane 5: HMW calibration kit.

Figure 5 MALDI-TOF-MS analysis of both PEGylated α-MMC and PEGylated MAP30 conjugates. (A) The (mPEG)₂-Lys-NHS was around 20 kDa and the PEG conjugates were observed at 49899.4 Da, 70430.9 Da and 91057.6 Da, reflecting 1, 2 or 3 PEG-bound chains per α-MMC molecule. (B) The (mPEG)₂-Lys-NHS was around 20 kDa and the PEG conjugates were observed at 50335.6 Da and 70614.2 Da, reflecting 1 or 2 PEG-bound chains per MAP30 molecule.

Figure 6 Topological inactivation activities. Lane 1: DNA marker; Lane 2: pUC18 DNA as a control; Lane 3 and 4: pUC18 DNA was incubated with α-MMC, PEG-α-MMC; Lane 5 and 6: pUC18 DNA was incubated with MAP30, PEG-MAP30.

Figure 7 Inhibitory effects of α-MMC/MAP30 and PEG-α-MMC/MAP30 on the proliferation of human choriocarcinoma JAR cells. JAR cells were treated with increasing concentrations for 72 hours. (A) and for various time points at 1.5 mg/mL. (B) of native or PEGylated α-MMC/MAP30 as indicated.

Note: Each data point represents the average of three independent experiments performed in quadruplicate.

Figure 8 Morphological differences in JAR cells for 72 hours culture photographed by phase contrast microscopy. JAR cells treated without drugs (A), with 15 μg/mL 5-fluorouracil (B), with 0.3 (C) and 1.5 mg/mL α-MMC (D) and with 0.3 (E) and 1.5 mg/mL PEG-α-MMC. (F) The white bar presents 50 μm.

Table 2 Herpes simplex virus-1-induced cytopathic effect (CPE) of VERO cell (50% tissue culture infective dose (TCID₅₀) = antilog [(55.6–50)/55.6–20) × (−1) + (lg10⁻⁵)] = 10^−5.2/0.1 mL = 10^−6.2/mL)

Virus ratio dilution	CPE appearing in cell holes		Total number of cell holes	CPE appearing in cell holes (%)
	Positive	Negative
−3	16	0	16	100
−4	10	1	11	90.9
−5	5	4	9	55.6
−6	2	8	10	20
−7	0	14	14	0
−8	0	20	20	0

Table 3 Inhibitory effects of RIPs and PEGylated RIPs on herpes simplex virus-1 (HSV-1) antigen

Protein	Concentration	OD450 of HSV-1 antigen	Inhibition ratio of HSV-1 antigen (%)	50% inhibiting concentration (μmol^−1)
Positive control	μmol · L^−1,*	0.923 ± 0.008	0
Negative control		0.004 ± 0.001
α-MMC	3.3	0.270 ± 0.011	71.055	0.67
	0.3	0.550 ± 0.009	40.588
	0.03	0.827 ± 0.003	10.446
	0.003	0.892 ± 0.004	3.373
α-MMC-PEG	3.3	0.452 ± 0.004	51.270	2.94
	0.3	0.689 ± 0.004	25.508
	0.03	0.866 ± 0.004	6.241
	0.003	0.902 ± 0.003	2.250
MAP30	3.3	0.220 ± 0.002	76.488	0.47
	0.3	0.507 ± 0.010	45.320
	0.03	0.807 ± 0.006	12.591
	0.003	0.890 ± 0.001	3.556
MAP30-PEG	3.3	0.429 ± 0.003	53.740	1.17
	0.3	0.674 ± 0.004	27.105
	0.03	0.854 ± 0.003	7.5472
	0.003	0.902 ± 0.000	2.286
Acyclovir	10	0.354 ± 0.004	61.901	2.39
	1	0.597 ± 0.006	35.450
	0.1	0.872 ± 0.003	5.515
	0.01	0.921 ± 0.006	0.254

Note:

* Compared with positive control P < 0.01.

Abbreviations: α-MMC, alpha-momorcharin; MAP30, momordica anti-HIV protein; PEG, polyethylene glycol.

Figure 9 Different concentrations of proteins on cell viability of VERO cells.

Abbreviations: α-MMC, alpha-momorcharin; MAP30, momordica anti-HIV protein; PEG, polyethylene glycol; ACV, acyclovir.

Figure 10 The ratio of cell viability under different concentration of proteins.

LowryOHRosebroughNJFarrALRandallRJProtein measurement with the Folin phenol reagentJ Biol Chem1951193126527514907713

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