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Purification of Glyoxalase-i and Catalase

Dr. Md. Motiar Rohman
Senior Scientific Officer
Plan Breeding Division, BARI, Gazipur

Abstract/ Executive Summary:

Glyoxalase-I (Gly-I) being important detoxifier of methyl glyoxal (MG) and Catalase (CAT) being the strongest decomposer of oxygen reactive species (ROS), this study was designed to purify very active Gly-I and CAT. Last year Gly-I and CAT activity were compared in 39 crops and among them onion showed the highest Gly-I activity (1.60 μmol/min/mg protein) followed by BARI sarisha-16 (1.33μmol/min/mg protein). In case of CAT, high activity was found in seedlings onion (303.3 μmol/min/mg protein), barley (199.2 μmol/min/mg protein) and BARI sarisha 16 (192 μmol/min/mg protein). Since CAT is the strongest decomposer of H₂O₂ and barley shows moderately tolerance to salinity, barley seedlings were subjected to isolate and purify. However, the recovery was very poor. On the other hand, Onion gly-I eluted with adjacent two bands.

Keywords: Purification, Glyoxalase-I, Catalase, Maize

Location: Planr Breeding Division, BARI, Gazipur

Start Date:

End Date:

Program Area: Variety and Species

Non-commodity: Maize

Objectives:

To purify CAT and Gly-I in molecular breeding lab.

Methodology:

Plant Materials: Maize (Zea mays L.)- (BHM 7); Onion (Allium cepa L.)-BARI piaz-3, barely (Hordeum vulgare L.)- (BARI barley 3).Extraction of crude protein for CAT purification: One hundred fifty gram onion bulb tissue and one hundred gram of 7 day old seedlings were homogenized in an equal volume of 25 mM Tris-HCl buffer (pH 8.0) containing 1mM EDTA 1% (w/v) ascorbate and 10% (w/v) glycerol with Waring blender. The homogenates squeezed in a nylon cloth and was centrifuged at 11500 x g for 15 min, and the supernatant was used as crude enzyme solution.Purification of Gly-I and CAT: For Gly-I purification DEAE and affinity chromatography and for CAT DEAE, Hydroxyl appatite-1,2 and Phynyl sepharose 4CL-B were used.Enzyme assay and protein quantification: Gly-I and CAT activities were assayed following the spectrophotometrically (Shimadzu, UV-1800). Gly-I and CAT activities were calculated using the extinction coefficient of 9.6 mM^-1cm^-1 39.4 M^-1cm^-1. Protein concentration was estimated following the method of Bradford (1976) using BSA as protein standard.SDS-PAGE, Silver staining and CBB dyeing: SDS-PAGE was done in 12.5% (w/v) followed by western blotting. Silver staining and CBB staining were done following standard methods.

Source of Information: Annual Research Report 2013-14, Plant Breeding Division, BARI, page: 163-164

Article Link (Online Journal):

Funding Source:

1. Government

Budget: One lac

Total Budget (Tk.) : One lac

Achievement/Findings:

Glyoxalase-I (Gly-I) being important detoxifier of methyl glyoxal (MG) and Catalase (CAT) being the strongest decomposer of oxygen reactive species (ROS), this study was designed to purify very active Gly-I and CAT. Last year Gly-I and CAT activity were compared in 39 crops and among them onion showed the highest Gly-I activity (1.60 μmol/min/mg protein) followed by BARI sarisha-16 (1.33μmol/min/mg protein). In case of CAT, high activity was found in seedlings onion (303.3 μmol/min/mg protein), barley (199.2 μmol/min/mg protein) and BARI sarisha 16 (192 μmol/min/mg protein). Since CAT is the strongest decomposer of H₂O₂ and barley shows moderately tolerance to salinity, barley seedlings were subjected to isolate and purify. However, the recovery was very poor. On the other hand, Onion gly-I eluted with adjacent two bands.

Knowledge Management: Report/Proceedings