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Purification of Glyoxalase-i from Maize (zea Mays L.)

M.M. Rohman
Plant Breeding Division, BARI

Abstract/ Executive Summary:

Since Glyoxalase-I (Gly-I) is important detoxifier of methyl glyoxal (MG), this study was designed to examine the level of Gly-I in maize and to purify it from maize seedlings. Both green and non-green part of maize seedlings were used to purify. Crude proteins were precipitated by 65% (NH₄)₂SO₄, and dialyzed over night. The dialyzate was applied on DEAE-cellulose chromatography and eluted with linear grade of KCl from 0 to 0.2 M. In both cases, Gly-I eluted at approximately 85 mM of KCL. The active Gly-I fractions were pooled and applied on affinity chromatography (S-hexyl glutathione-agarose) and eluted with 1.2 mm of S-hexyl glutathione. The purified protein from green and non-green part had specific activity of 33.23 and 29.25 μmol min^-1 mg^-1 protein, respectively along with recovery of 1.47 and 162, respectively and yield of 83.11 and 68.15, respectively. In SDS-PAGE, the active purified affinity fraction was found to move with another protein. The spectrophotometric analysis showed the the Gly-I protein elutes with GST protein.

Keywords: Glyoxalase, Spectrophotometric analysis, Detoxifier, Maize

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Maize

Objectives:

To purify Glyoxalase-i from maize seedlings considering the importance of glyoxalase system in MG detoxification

Methodology:

The green part (leaf) and non-green part (except leaf) of 7 day old maize seedlings were as plant materials. Fifty gram green tissue were homogenized in an equal volume of 25 mM Tris-HCl bu?er (pH 8.0) containing 1 mM 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. Proteins were precipitated by ammonium sulfate at 65% saturation from the supernatant and centrifuged at 11,500 × g for 10 minutes. The proteins were dialyzed against 10 mM Tris-HCl buffer (pH 8) containing 0.01% (w/v) β-mercaptoethanol and 1 mM EDTA (buffer A) overnight to completely remove low molecular inhibitors. The dialyzate was applied to a column (1.77 cm i.d. × 20 cm) of DEAE-cellulose (DE-52, Whatman, UK) that had been equilibrated with buffer A and eluted with a linear gradient of 0 to 0.2 M KCl in 600 ml of buffer A. The active eluted Gly-Ipeak was collected for further purification.The high active pooled sample of Gly-I separated by DEAE-cellulose column chromatography, was applied on a hydroxyapatite column (1.5 cm i.d. × 5.5 cm) that had been equilibrated with buffer A. The column was eluted with a 300 ml linear gradient of potassium phosphate buffer (K-P buffer; 0-40 mM, pH 7.0) in buffer A. The high active fractions were collected and further purified by affinity chromatography.The collected sample was applied to a column (0.76 cmi.d. × 4.0 cm) of S-hexyl glutathione-agarose that had been equilibrated with 10 mM Tris-HCl buffer (pH 8.0) containing 0.01% (v/v) β-mercaptoethanol (buffer B). The column was washed with buffer B containing 0.2 M KCl and eluted with buffer B containing 1.2 mM S-hexyl glutathione. The high active protein fractions eluted with S-hexyl glutathione were combined and dialyzed against buffer B, and the dialyze was used as the purified Gly-I. The activity and absorbance (A₂₈₀) were taken.Gly-I activity was assayed following the methods of Hossain et al. (2007), respectively, spectrophotometrically (Shimadzu, UV-1800) and were calculated using the extinction coefficient of 3.37 mM^-1cm^-1. Protein concentration was estimated following the method of Bradford (1976) using BSA as protein standard. To check the homogeneity of purified GST enzyme and to estimate its molecular mass, SDS-PAGE was done in 12.5% (w/v) gel containing 0.1% (w/v) SDS by the method of Laemmli (1970) followed by silver staining.

Source of Information: Annual Research Report, Plant Breeding Division, BARI--2014-15

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Achievement/Findings:

In extracts of crude protein from green part and non-green part, it was found that the Gly-I had higher specific activity in protein extract from non-green part. During separation of protein by anion exchange chromatography, the Gly-I eluted at approximately 85 mM of KCl for both case. In DEAE-cellulose fraction from green and non-green part contained 6.43 and 8.73 μmol min^-1 mg^-1 protein activity, respectively. The pooled Gly-I fractions was applied of hydroxylapatite chromatography, but the elution lost the activity soonest. Therefore, the active Gly-I fractions were applied to affinity chromatography (S-hexyl glutathione-agarose) for final purification and eluted with 1.2 mM of S-hexyl glutathione. The purified protein from green and non-green part had specific activity of 33.23 and 29.25 μmol min^-1 mg^-1 protein, respectively along with recovery of 1.47 and 162, respectively and yield of 83.11 and 68.15, respectively. It was remarkable that the protein concentration in final product was very low. Therefore, the fraction of affinity chromatography was applied to examine the purity and concentration of Gly-I. In silver staing of SDS-PAGE, the active purified Gly-I fraction was found to move with another protein. Since in purification of Gly-I the chromatographical use, the additional protein band might be due to GST. Therefore, the fractions of DEAE-cellulose and affinity chromatography were subjected to examine GST activity. Considering this study along with previous results it could be concluded that Gly-I has similar physical properties. In DEAE, the GST activity started increasing under the Gly-I peak. Therefore, in affinity column, Gly-I and GST eluted in same fraction. In this connection, it could be suggested that in SDS-PAGE the additional protein band might be due to presence GST protein. Considering this study and previous results, Gly-I in maize not so responsive to abiotic stress. Therefore, it is to more constitutive rather than functional. In this context, genetic engineering of a high active stress inducible Gly-I might improve the tolerance under drought and other abiotic stress.

Knowledge Management: Report/Proceedings