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Antibacterial Activity of Trichosanthes Cucumerina Seed Lectin and Study of Its Structural Stability by Fluorescence Spectroscopy

Md. Golam Kibria
Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi-6205, Bangladesh

Md. Rezaul Karim
Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi-6205, Bangladesh

Imtiaj Hasan
Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi-6205, Bangladesh

A.K.M. Asaduzzaman
Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi-6205, Bangladesh

Md. Belal Uddin
Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi-6205, Bangladesh

Syed Rashel Kabir
Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi-6205, Bangladesh

Abstract/ Executive Summary:

A Trichosanthes cucumerina seed lectin (TCSL) was purified previously that showed potent inhibitory effects against Ehrlich ascites carcinoma (EAC) cells in vivo in mice. In the present study, the lectin was treated with guanidine-HCl for 2 and 4h in the presence and absence of Ca²⁺ and changes in the tryptophan fluorescence shift were monitored by fluorescence spectroscopy. It was found that the lectin stability was increased in the presence of Ca²⁺. Although the denaturant changed the environment of tryptophan residue, it did not affect the binding sites of TCSL as red blood cells became agglutinated after the treatment with EDTA. Besides the agglutination of three pathogenic bacterial species, the lectin also partially inhibited the growth of Salmonella enteritidis and Staphylococcus aureus.

Keywords: Fluorescence spectroscopy; Bacterial agglutination; Bacterial growth inhibition, Snake gourd

Location: Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi-6205, Bangladesh

Start Date:

End Date:

Program Area: Seed Technology

Commodity: Snake gourd

Objectives:

To observe the antitumor activity of TCSL, its structural stability, bacterial agglutination and antibacterial activity against the pathogenic bacteria was studied in this manuscript.

Methodology:

Purification of TCSL Lectin was purified from Snake gourd seeds and the protein concentration was determined by Lowry’s methods where BSA was used as the standard protein. Agglutination of red blood cells was studied. Bacterial Agglutination Activity Listeria monocytogenes, Salmonella enteritidis, Shigella flexneri, Staphylococcus aureus, Shigella boydii and Pseudomonas aeruginosa were used to examine the bacterial agglutination activities of TCSL. The bacteria were grown at 37°?C for 18 h in liquid nutrition medium and centrifuged at 1,027 g for 5 min and washed with 10 mM Tris-HCl buffer saline, pH 7.8. The bacteria were re-suspended in the same buffer with a turbidity of 2.3 at A₆₃₀. Then TCSL (0.8 mg/ml) was serially diluted with a hemagglutination buffer in the presence and absence of 0.8 mM of lactose and 50 µl of each bacterial suspension was mixed to a final volume of 100 µl in a 96-well microtiter plates. The plate was agitated for 2 min and was kept at room temperature for 60 min. Finally, light microscope was used to monitor the bacterial agglutinating activity. Bacterial Growth Inhibition In the presence and absence of different concentrations of TCSL (30 µg/ml - 240 µg/ml) in bacterial nutrient broth, the bacterial growth inhibition was studied according to the method previously described. Three species of bacteria (Pseudomonas aeruginosa, Salmonella enteritidis and Staphylococcus aureus) were used for this study. The bacteria were grown overnight in the nutrient broth at 37ºC and the absorbance was adjusted to 0.18-0.2 at A₆₃₀ by adding the liquid nutrient medium. Then TCSL was serially diluted in the nutrient broth in a 96-well microtiter plate and 50 µl of each bacterial suspension was mixed to a final volume of 100 µl. Four wells without the lectin for each bacterium were used as control. The reading was taken at A₆₃₀ after the plates were agitated for 8 h at 32°C by using temperature controlled titer plate shaker. According to the following formula the percentage of bacterial growth inhibition was determined: % inhibition = {(Absorbance of control - Absorbance of test) /Absorbance of control} × 100. Fluorescence spectroscopy- The fluorescence measurement of TCSL (40 and 50 µg/ml) in tris buffer saline (TBS) has been performed at 30°C temperature by using a Shimadzu Spectrofluorometer RF-5301 PC. Fluorescence spectrum of 40 µg/ml TCSL was taken in the presence and absence of 1mM of CaCl₂. TCSL (50 µg/ml) was treated with 0.5 M of Guanidine-HCl for 2 h and 4 h in the presence of 1 mM of CaCl₂. All samples were placed in a 1×1×4.5 cm quartz cuvette, excited at 280 nm and the emission was recorded in the range of 300-400 nm Widths for the excitation and emission monochromators were maintained at 5 nm.

Source of Information: International Journal of Biosciences | IJB |, Vol. 9, No. 6, p. 187-192, 2016

Article Link (Online Journal): http://dx.doi.org/10.12692/ijb/9.6.187-192

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

TCSL inhibited the growth of a number of pathogenic bacteria. Though the denaturant changed the environment around its tryptophan residues, binding sites of CSL did not become affected. The lectin stability increased in the presence of Ca²⁺ salt that would be very important for the formulation of the lectin as an antibacterial agent.

Knowledge Management: Journal