1. 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: Disruption of cells using Results:
homogenizer.
lactate dehydrogenase (LDH) is a 35 kda enzyme with a Figure 2 shows the results of the purification of
theoretical pI of 7.5 which catalyses the nicotinamide lactate dehydrogenase from chicken muscle by
cofactor-dependent inter conversion of lactate and affinity chromatography using Blue Sepharose
pyruvate. 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
Centrifugation ensuring debris samples and the enzymatic activity elutes with a
Removal
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
LDH is found in most of the organisms because it plays which was purified at this stage of chromatography
a vital role in carbohydrate metabolism. During and it confirms the presence of our protein after it
conditions in which pyruvate production from glycolysis was stained by Coomassie brilliant blue. (Figure 1).
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 Precipitation/concentration
using Ammonium Sulphate
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. Dialysis for salt Removal
Different analytical methods were employed to 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
determine the presence and purity of LDH enzyme. purified LDH protein band obtained after stepwise gradient elution fractions from
Blue sepharose column (1M NaCl in 10 mM Tris-HCl, pH 7.4).
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 Purification through blue
sepharose affinity
by using homogenizer and centrifuge respectively chromatography.
followed by precipitating the proteins at 35% and 70 %
Figure 2: Blue Sepharose affinity chromatography :A 70% Ammonium sulphate cut
ammonium sulphate fractionation and removal of of the crude extract was subjected to Dialysis for removal of excess salts and ,
excess salts from the Dialysis method. The purification 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
of the protein is performed by affinity activity and SDS-PAGE analysis.
chromatography.Then our desired protein binds
strongly to the column and eluted as stepwise gradient References:
1.Alcazar O, Tiedge M, Lenzen S ,2000 ,Importance of lactate dehydrogenase for the
elution at later stages of chromatography. In this lab regulation of glycolytic flux and insulin secretion in insulin-producing cells. Biochem
we used a blue Sepharose affinity column to purify J352:373–380.
2.Allen R. Place and Dennis A. Powers ,1984, Purification and Characterization of the
LDH,and subsequently purity and concentration of LDH Analyze Purified Protein through
Lactate Dehydrogenase (LDH-B4) Allozymes of Fundulus heterocZitus,JBC, Vol. 259, No. 2,
SDS PAGE.
is determined using activity assays and SDS-PAGE. 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.