Proteomic Strategies for purification of lactate dehydrogenase

Gaurav Dwivedi,Hareesha Kakkera

Department of Chemistry Umeå University, SE-901 87 Umeå, Sweden

Contact: gadu0002@student.umu.se

Background:

lactate dehydrogenase (LDH) is a 35 kda enzyme with a theoretical pI of 7.5 which catalyses the nicotinamide cofactor-dependent inter conversion of lactate and pyruvate.

LDH is found in most of the organisms because it plays a vital role in carbohydrate metabolism. During conditions in which pyruvate production from glycolysis exceeds the ability of the cell to metabolize the pyruvate, LDH converts the pyruvate to lactate, and thus regenerates the oxidized NAD required for further glycolysis. LDH also allows the conversion of lactate to pyruvate.

Aim:

The purpose of this study is to extract and purify LDH enzyme from chicken muscle using a variety of techniques including centrifugation, selective protein precipitation, dialysis and affinity chromatography. Different analytical methods were employed to determine the presence and purity of LDH enzyme.

Methods:

Extraction of LDH enzyme from chicken muscle is achieved by placing frozen chicken cubes (50 gm) in 100 ml of extraction buffer (20mM Tris-HCl, pH 8.6) ,homogenization and removal of the cell debris is done by using homogenizer and centrifuge respectively followed by precipitating the proteins at 35% and 70 % ammonium sulphate fractionation and removal of excess salts from the Dialysis method. The purification of the protein is performed by affinity chromatography.Then our desired protein binds strongly to the column and eluted as stepwise gradient elution at later stages of chromatography. In this lab we used a blue Sepharose affinity column to purify LDH,and subsequently purity and concentration of LDH is determined using activity assays and SDS-PAGE.

Figure 1:SDS –PAGE profiling: SDS-PAGE pattern of protein fractions obtained during purification of LDH from chicken heart muscle. Arrow indicates the position of purified LDH protein band obtained after stepwise gradient elution fractions from Blue sepharose column (1M NaCl in 10 mM Tris-HCl, pH 7.4).

Figure 2: Blue Sepharose affinity chromatography :A 70% Ammonium sulphate cut of the crude extract was subjected to Dialysis for removal of excess salts and , applied to a Blue Sepharose column, then eluted with a stepwise gradient elution fractions as described in Methods. The fractions collected were assayed for LDH activity and SDS-PAGE analysis.

Disruption of cells using homogenizer.

Centrifugation ensuring debris Removal

Precipitation/concentration using Ammonium Sulphate

Dialysis for salt Removal

Purification through blue sepharose affinity chromatography.

Analyze Purified Protein through SDS PAGE.

Results:

Figure 2 shows the results of the purification of lactate dehydrogenase from chicken muscle by affinity chromatography using Blue Sepharose column. The success of the chromatographic purification is illustrated in the same figure 2.The bulk of the protein is eluted from the column where a peak was shown corresponding to the flow through samples and the enzymatic activity elutes with a smaller protein peak half way through the elution profile. SDS-PAGE profiling of this protein peak from the elution sample shows the desired protein band which was purified at this stage of chromatography and it confirms the presence of our protein after it was stained by Coomassie brilliant blue. (Figure 1).

References:

1.Alcazar O, Tiedge M, Lenzen S ,2000 ,Importance of lactate dehydrogenase for the regulation of glycolytic flux and insulin secretion in insulin-producing cells. Biochem J352:373–380.

2.Allen R. Place and Dennis A. Powers ,1984, Purification and Characterization of the Lactate Dehydrogenase (LDH-B4) Allozymes of Fundulus heterocZitus,JBC, Vol. 259, No. 2, pp. 1299-1308.

3.Hultin, H. O., and J. H. Southard. 1967. Cellular distribution of lactate dehydrogenase in chicken breast muscle. J. Food Sci.32:503–510.

Acknowledgments: Special thanks and recognition to Aaron Edwin and Moritz Müller our lab supervisors and Karina Persson coordinator for the Program at Dept of Chemistry, Umeå university.