Hemagglutinating activity of proteins from Parkia speciosa seeds

Figures & data

Table 1.  Hemagglutinating activities and yields at various stages of purification of lectin from Parkia speciosa.

Chromatographic fraction	Activity		Yield (μg)
	Hemagglutinating activity with rabbit erythrocytes (IC50 μg/μL)	α-Glucosidase inhibition (% inhibition)
Crude 25%	6	—	1.19 × 10^5
A1	4	55	2.4 × 10^3
Gj	0.39	—	31.02
Crude 60%	2.51	5	1.29 × 10^6
C2	1.17	—	1.35 × 10^3

Note. —, no activity.

Figure 1.  Affinity chromatogram of crude protein of Parkia speciosa on Affi-Gel Blue gel column (1.6 × 10 cm) in binding buffer 1 mM Tris-HCl, pH 7.2. Dashed lines indicate use of 0–100% linear gradient of 0.5 mM NaCl in binding buffer. Flow rate 1.5 mL/min.

Figure 2.  Gel filtration chromatogram of A1, A2 fractions from Affi-Gel Blue gel chromatography on Superdex 200 column (1.6 × 60 cm) in double distilled water. Flow rate 0.5 mL/min.

Figure 3.  MALDI-ToF mass spectrum of Gj fraction from Superdex 200 gel filtration.

Table 2.  Proteins identified by MALDI-ToF mass spectrometry followed by peptide mass fingerprinting via the Mascot search engine.

Fraction no.	Protein	Database accession number	MW (kDa)	Theory MW	Organism	Score*	% Protein covered
Gj	Putative aristolochene synthase, 3’-partial	Q8LN52_ORYSA	21.08	23,232	Oryza sativa	72	41
C2a	protein B1342F01.11	Q69JW1_ORYSA	45	14,184	Oryza sativa	115	55
C2b	Hypothetical protein At1g51560	Q8VXY3_ARATH	23	43,571	Arabidopsis thaliana	91	35

Note. *Probability based Mowse score: protein score is–10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 78 are significant (p < 0.05).

Figure 4.  Affinity chromatogram of crude protein from Parkia speciosa on Con A Sepharose column (1.6 × 5 cm). Flow rate 1.5 mL/min. The equilibration buffer was 20 mM Tris-HCl, pH 7.4, containing 0.5 M NaCl, 1 mM CaCl₂·2H₂O, 1 mM MnCl₂. The eluted lectin buffer was 20 mM Tris-HCl, pH 7.4, containing 0.1–0.5 M methyl-α-d-mannopyranoside.

Figure 5.  One-dimensional SDS-PAGE of lectin from Parkia speciosa (C2a and C2b) was visualized with Coomassie staining. 12.5% acrylamide gel was used.