GRANT APPLICATION

Title of the grant:Purification of lactate dehydrogenase from chicken breast muscule.

Applicants:GauravDuttadwivedi andHareeshaKakkera.

Organization: Department of Chemistry, Umeå University, Linnaeus Väg 6 (KBC huset), 90187

Umeå, Sweden.

Aim of the project:

The purpose of this experiment was to extract and purify LDH enzyme from chicken breast muscle using a variety of techniques including centrifugation, selective protein precipitation, and dialysis and 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 purification of LDH is essential for studying the function, itsstructure, and interactions of this protein.

Introduction and Background:

Lactate Dehydrogenase (LDH) is a glycolytic enzyme which catalyzes the change of pyruvate and

NADH to lactic acid and NAD+, or vice versa. The purpose of this pathway is to regenerate NAD+,

which can be reduced to NADH in the early steps of glycolysis. This pathway is important for

generating ATP in the absence of oxygen, and is a primary source of NAD+ for anaerobic cells that

rely on fermentation for energy.

In most animal tissues, LDH is produced from two genes, designated A and B. The A gene is

somewhat more highly expressed in muscle and liver, and its product is referred to as the M isozyme,

while the B gene is more highly expressed in heart, and its gene product is referred to as the H

isozyme.

LDH activity is readily measurable: the extinction coefficient for NADH at 340 nm (6220 (M•cm)-1) is

much higher than that of NAD (the ε340 for NAD is zero). If the only substrates added to the reaction

are NAD and lactate (or NADH and pyruvate), the change in absorbance at 340 nm should be

proportional to the change in NADH concentration due to the LDH activity present in the cuvette.

Most tissues contain proteases (enzymes that degrade other proteins). Avoiding proteolytic damage

to your protein can be difficult. Three techniques are commonly used to keep proteolysis to a

minimum: 1) perform the purification in the presence of protease inhibitors, 2) perform the

purification at low temperatures (4°C or on ice), and 3) perform the purification in the minimal

amount of time possible.

Experiment/Work plan

To purify the LDH, protein, westarted 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. If the protein is in solution, we expose it to a selective precipitation. Selective precipitation of proteins can be used as a rough method to recover a desired protein in purification. 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 is a process of sorting out molecules in solution by differences in rates of diffusion across a semi-permeable membrane. Dialysis can most often remove a large amount of small impurities in a heterogeneous solution containing your protein. We used dialysis in this lab toremove the excess, unwanted (NH4)2SO4 and other small impurities while simultaneously exchanging the extraction buffer with dialysis buffer. Affinity chromatography is used to obtain a specific substance if it’s mixed in aheterogeneous solution. Columns used for affinity chromatography are typically composed if inert, chemically stable polymers that have specific binding proteins or molecule. Weused a column that was composed of a specific molecule to which the protein of interest would bind to with a high affinity. Once the protein solution is applied to the column, all substances and proteins that do not bind or that bind loosely to the column are removed with buffer washes. Then the column is washed with a solution to which the desired protein binds strongly to and is isolated. 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. To obtain pure LDH from the column, it was washed with an NADH solution because of the high affinity LDH has for NADH. Once our protein is purified, there are many techniques to determine the purity and Concentration of your protein. First we typically run an activity assay if the protein has enzymatic properties, 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.

Future plans

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

Expression of Lactate Dehydrogenase in E.coli using recombinant DNA technology and use of various “tags”.

ExecuteColorimetric Assays for protein determination. Perform Gel filtration chromatography.

Long term goal

Understanding the interactions with its substrates by studying the enzyme kinetics. Carrying out protein crystallization to unravel the 3 D structure and relate its function. Analyze the effects of an inhibitor, and studying the effects of chemical modification of the

enzyme.

Budget

Institution Name:Umeå University Principal Investigator(s):GauravDutta dwivedi &HareeshaKakkera. Project Title:Purification of lactate dehydrogenase from chicken breast muscule. Grant Period: from 09-0 5-2011 to 09-05-2014

Year 1 (2011) Year 2 (2012) Year 3 (2014) Total

Personnel

Salaries

Principal Investigator $11,200 $22,400 $11,200 $44,800

Co-PI(s) $11,200 $22,400 $11,200 $44,800

Research Assistant $32,000 $64,000 $32,000 $128,000

Staff $5,000 $10,000 $5,000 $20,000

Tuition/Fees

Benefits

Subtotal Personnel $59,400 $118,800 $59,400 $237,600

Project Expenses

Fees/Stipends

Supplies

Communication $10,000 $10,000

Transcription $8,000 $8,000

Equipment

Travel $6,000 $15,000 $21,000

Miscellaneous $12,000 $12,000

Subtotal Project Exp. $18,000 $23,000 $10,000 $51,000

Total Direct Costs $77,400 $141,800 $69,400 $288,600

Indirect Costs (15%) $11,610 $21,270 $10,410 $43,290

Sub-Contract(s)

Total Project Costs $89,010 $163,070 $79,810 $331,890