grant application
TRANSCRIPT
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