Purification of lactate dehydrogenase (LDH) from chicken muscle

Gaurav Dutta DwivediHareesha Kakkera

Aim of the project

• The purpose of this experiment is to extract and purify LDH enzyme from chicken muscle using appropriate protein purification techniques.

Strategy applied for the purification process

Techniques applied for achieving this: • Centrifugation.• Selective protein precipitation.• Dialysis.• Affinity chromatography.Many different analytical methods were employed to determine the presence, purity and concentration of LDH such as activity assays and SDS-PAGE.

The Protein:lactate dehydrogenase

In this course, we used lactate dehydrogenase (LDH) as the subject of our studies.It is an vital player in the anaerobic generation of energy from glucose as well as in the synthesis of glucose from lactate. It carry out the following reaction:

Lactate + NAD+ LDH Pyruvate + NADH + H+

Enzyme clears lactic acid from working musclesThe obvious source of enzyme is muscle tissue (heart & skeletal muscle, H&M, isomers)

We detect in assay for the enzymes ability to convert Pyruvate to Lactate.

Brief overview of the experiment• Disrupt

– Blender, homoginizer • Remove debris

– Centrifugation• Precipitate/concentrate

– Ammonium sulfate• Remove salt

– Dialysis• Purify

– Chromatography• Analyze

– Activity, molecular weight

Disrupt the cells

• We must start with a tissue sample containing the protein.

• Most proteins are typically found within a cell, so the tissue must be subjected to a homogenizing process in order to break cell walls and release protein.

Remove debris• Centrifugation of this extract

at moderately high speeds sediments the nuclei, mitochondria, membranes and other insoluble material.

• The remainder of the purification is an attempt to separate the LDH contained in the clear supernatant from the other soluble proteins.

• The large centrifuge tube must then be balanced before it can be placed in the instrument; this is done using a separate tube filled with water and a pan balance.

Ammonium Sulfate ppt

• One of the most commonly used crude purification techniques involves the use of differential solubility.

• Proteins precipitate with increasing ammonium sulfate concentrations, with most proteins precipitating somewhere between 10% and 60% ammonium sulfate.

• This process depends on the physical or chemical interaction between the protein and the precipitating agent. In this lab, (NH4)2SO4 is used to precipitate out LDH.

Dialysis

• Passage of solutes through a semi-permeable membrane.

• Pores in the dialysis membrane are of a certain size.

• Protein stays in; water, salts, protein fragments, and other molecules smaller than the pore size pass through.

• We used dialysis in this lab to remove the excess, unwanted (NH4)2SO4 and other small impurities while simultaneously exchanging the extraction buffer with dialysis buffer.

Affinity chromatography

• Most purification methods involve chromatography. Chromatographic methods involve a column of an insoluble material that can bind molecules based on specific properties common to proteins.

• The solution containing the mixture of proteins is then allowed to pass through the column; the protein of interest may bind (depending on its properties), while at least some impurities remain in solution and leave the column. The procedure is completed by eluting (i.e. “removing”) the proteins that have bound to the column.

• In this lab we used a Cibacron blue affinity column to purify LDH. This molecule mimics the shape and charge characteristics of pyridine nucleotides to which dehydrogenase proteins frequently bind to.

Analyzing the purity by SDS-PAGE gel

• To determine which fractions from the chromatography contain the protein. SDS-PAGE gel electrophoresis is a good method to use for determining the purity of the protein.

• This method separates proteins according their molecular weight and length of polypeptide chain. Proteins all exhibit the same charge per unit mass due to the binding of SDS resulting in fractionation by size and mass.

SDS PAGE for Purification

Result of the SDS-PAGE

Purified protein band

Future strategies

• Performing a Western Blot analysis to confirm the presence of LDH in each sample and fraction.

• Expression of Lactate Dehydrogenase using recombinant DNA technology and use of various “tags”.

• Execute Colorimetric Assays for protein determination.

• Perform Gel filtration chromatography.

Long term objectives

• Recognize the interactions with its substrates by studying the enzyme kinetics.

• Perform protein crystallization to unravel the 3 D structure and relate its function and structure.

• Analyze the effects of an inhibitor, and studying the effects of chemical modification of the enzyme.

 

Application of lactate dehydrogenase

• LDH is often used as a marker of tissue breakdown, therefore it can be used in detection of  myocardial infarction, hemolysis.

• Variation in LDH isozymes profile might have a role in studying muscle response to training so its used in sports medicine.

Thank you !