Lactate Dehydrogenase Lab Report

Lactate Dehydrogenase (LDH) is an cytosolic enzyme that participates in anaerobic glycolysis. LDH couples the reduction of pyruvate into lactic acid to the oxidation of NADH to NAD+, which allows glycolysis to continue to produce ATP in the absence of oxygen. LDH is a tetramer, a protein complex of 4 polypeptide subunits, composed either a subunit expressed strongly in the heart, H type,or a subunit that is highly expressed in the muscle, M type. There are 5 isozymes of LDH composed the 2 subunit types. The molecular weight of each subunit is approximately 36.6 kDa and a total of 146 kDa for the enzyme. The isoelectric point of the H type is 5 and the M type subunit is 8. Using the different pI of these isozymes, LDH determined the identify
of the pig tissue from which the enzyme was purified.
Proteins in the crude homogenate may differ in cellular localization, molecular weight, hydrophobicity, ionic interactions, and substrate affinity. The first characteristic that was separated for was cellular localization. The cytosol was isolated from the crude homogenate using a series of centrifuges. Salt concentrations disrupt the solubility of protein in solution for further purification of LDH from more hydrophobic cytosolic proteins. LDH was precipitated in a 65% cut of ammonium sulfide, spun down and resuspended for a Cibacron Blue ligand Sepharose resin Affinity Chromatography. The Cibacron Blue mimics the Adenine base ring of NAD+ but has a higher dissociation constant than NAD+ for simple elution. Low concentration of NaCl increases the dissociation constant of Cibacron Blue and the substrate is eluted at a faster rate than NAD+. The salt disruption of NAD+ affects the intermolecular charge interaction before significant LDH can be eluted. The ligand bound LDH, along with other nucleotide affinity enzymes, to the sepharose resin while proteins with no affinity for the ligand were eluted from the column. Size Exclusion separates based on the molecular weight of protein. Proteins of similar biochemical identities as well as molecular weight as LDH, which is a small fraction of total protein is the final product. A spot test for LDH activity using the reaction ............. was used to determine the fractions that contained LDH. purified with a Size-Exclusion Chromatography.

The first step in purifying LDH is to open up cellular compartment with blending in a buffer to make the crude homogenate. A series of centrifuge and decanting the supernatant was done to remove dense proteins, large organelles, and debris from the crude homogenate. After decanting the clarified homogenate, which should contain soluble organelles and the cytosol, 40% cut of ammonium sulfide and precipitated. The ammonium sulfate dissociates and interacts with water disrupting the water shell around the protein that allows the protein to remain soluble in solution. Increasingly less hydrophobic proteins are precipitated with the higher cut of ammonium sulfide. Ammonium sulfate was added to the supernatant to increase cut to 65%. Proteins precipitated were centrifuged and resuspended in buffer.
LDH has an affinity towards NAD+, being one of its substrates, thus an affinity chromatography was run with resin that have ligand that is an analog of the adenosine. To determine when all of unbound proteins have run through the column, absorbance readings were taken periodically to create a chromatogram. . The bound protein is dissociated with the ligand with a washing of salt which disturbs the charge interaction between the substrate and enzyme. (where I explain how we find the fraction where LDH can be found)
The only difference between proteins now is the molecular weight. However before there can be g https://www.jstage.jst.go.jp/article/biochemistry1922/107/1/107_1_138/_article http://www.ncbi.nlm.nih.gov/pubmed/1637926 http://www.ncbi.nlm.nih.gov/pubmed/7670598 http://www.ncbi.nlm.nih.gov/pubmed/21526780 http://www.ncbi.nlm.nih.gov/pubmed/20951115 Method
Isolation of Cytosolic Proteins
100 mL of unknown crude homogenate was centrifuged at 20,000 xg for 10 minutes at 4 celsius.
The supernatant was decanted into a clean pre-chilled beaker and the pellet was discarded. A 0.5 mL sample was frozen down for later analysis of purification method. The clarified homogenate was rapidly stirred with a pre-chilled stir bar while 19.77 grams of ammonium sulfate was added slowly for a 40% cut precipitation. The clarified homogenate was stirred for 15 minutes after the ammonium sulfate was dissolved. The clarified homogenate was centrifuged for 15 minutes at 15,000 gx. The volume of the 40% cut supernatant was measured and the pellet was discarded. The 40% cut was stirred rapidly while 14.61 grams of ammonium sulfate was slowly added for a 65% cut precipitate and was stirred slowly for an additional 15 minutes. The 65% cut was centrifuged for 15 minutes at 15,000 xg. The supernatant was discarded. The pellet was resuspended in cold 4 mL of pH 7.5 phosphate buffer and transferred to a pre-chilled test tube for further purification. 200 uL was frozen down for later
analysis.
An affinity chromatography was prepared in a 1 cm diameter by 10 cm length column and loaded with 6 mL of Cibacron Blue-agarose resin. The column was equilibrated with 12 mL of phosphate buffer. Before loading the 65% cut pellet into the column, a 20 uL sample was taken as the positive control for later spot test. The 65% cut pellet was centrifuged at 5,000 RPM for 5 minutes at 4 celsius. The supernatant was decanted into a clean test tube and was warmed to room temperature. The pellet was discarded. When the 65% cut pellet was loaded, the column was washed with phosphate buffer and the drop rate was approximately 1 drop for every 10 seconds. The eluted volume of 2, 4, 6, 8, 11, 14, 17, 20, 23, and 26 mL were gathered in a cuvette and absorbance measurement was taken to track the elution of unbound protein. When 27 mL of volume was eluted, the column was washed with 20 mL of 1 M NaCl. 20 microfuge tubes were labeled 1-20 and were filled with 1 mL of the respective fraction. Absorbance measurements were taken for every other fraction. The column was washed with 2 M of NaCl when the last fraction was collected. To identify the fractions that contained LDH was spot test that tests for NAD+ to NADH reduction with the oxidation of PMS and the subsequent reduction of nitro-blue tetrazolium. When nitro-blue tetrazolium is reduced the well becomes blue and indicates the presence of LDH. A regent mix was prepared with 2.16 mL of 25 mM lactate in 100 mM CAPS buffer, 240 uL of 10 mM NAD+, 60 uL of 2 mM NBT, and 15.6 mL of 2 mM of PMS. 100 uL of reagent mix was added to 22 wells. 2 ul of 65% pellet taken before loading into the column was loaded into the positive control well and nothing was added to the negative control well. Each well was then loaded with 2 uL of an affinity chromatography fractions. The wells were developed for 5 minutes. Fractions that turned blue were combined in a pre-chilled beaker with a stirring bar and 5.41 grams of ammonium sulfate was added with rapid stirring. The volume of the combined fractions was then measured.
The size exclusion column was prepared a week in advance of running the column for tight packing of the resin beads. The column was loaded with with 22 mL of Sephadex G100.
The Affinity purified LDH fraction was centrifuged at 15,000 xg for 15 minutes at 4 celsius. The supernatant was discarded. The pellet was resuspended in 150 uL of cold phosphate buffer. The volume of the Affinity pellet was recorded and two separate 20 uL samples were frozen down for later analysis. 200 uL of Blue Dextran and 100 uL of Orange-G was added to the Affinity purified pellet to track the elution of the sample. The sample was warmed to room temperature before being loaded into the column. 20 microfuge tubes were numbered 1 through 20 and a mark for 1 mL was labeled for each.
The column was washed with 5 mL of phosphate buffer. The rate of elution was set at one drop per 13 seconds. When the meniscus reached the packed beads, the sample was loaded. Fractions were collected in labeled microfuge tubes. A spot test that was used to identify the LDH fraction of the Affinity Chromatography was repeated to identify the presence of LDH in Size Exclusion fractions. Fractions that appeared to have a dark blue reaction were combined. 6.49 grams of ammonium sulfate was mixed into the combined fractions and sat on ice for an additional 15 minutes. The mixture was centrifuged at 15,000 xg for 15 minutes at 4 celsius. The supernatant was discarded and the pellet was resuspended in 1 mL of phosphate buffer. The volume of this pellet was taken. To analyze the effectiveness of the purification method LDH Activity Assay and Bradford Assay were performed using samples that were collected from the purification intermediates. For both Assays the samples were diluted. 100 uL of 1:10 diluted Clarified Homogenate, 100 uL of 1:50 diluted 65% cut, 100 uL of 1:1250 diluted Affinity Purified LDH, and 100 uL of 1:50 diluted Size Exclusion purified LDH were made. The LDH Activity Assay measured the increase in Absorbance as NAD+, which has no absorbance, is converted to NADH, which peaks at 340. The Spectrophotometer was set to measure Absorbance at 340 nm. A master mix equivalent for 10 assays was made by mixing 1 mL of 10 mM NAD+ and 9 mL of 100 mM CAPS/25 mM lactate stock. 5 cuvettes were loaded with 1 mL of master mix. The blank had nothing added to the master mix. The four other cuvettes had 2 uL of an intermediate loaded and inverted twice before being loaded into the Spectrophotometer. The Bradford Assay used the same diluted intermediate samples. The Spectrophotometer was set to measure Absorbance at 595 nm. 15 test tubes were labeled and loaded with 900 uL of Bradford reagent. After preparing the LDH samples, the samples were vortexed, sat for 5 minutes, and transferred to a cuvette to record Absorbance. The subunit that the LDH was composed was identified using an electrophoresis gel. The Gel was prepared by heating 50 mL of 1% agarose in 1X Tris-Glycine running buffer. The Tris-Glycine Runner Buffer was composed of 20 mM tris, 20 mM glycine, and 2 mM EDTA at the pH 9.5. The electrophoresis unit was set up and the agarose was loaded and cooled for 25 minutes. The LDH sample was prepared by adding 1 unit of the Size Exclusion purified LDH, which was found to be 16 uL, 4 uL of 5X native gel sample buffer. The sample buffer contained 0.2 M tris, 0.2M glycine, 10 mM EDTA, 0.06% bromophenol blue, and 25% glycerol at a pH of 8.8. The gel ran at 80V for 1 hour and 30 minutes. The Gel was removed from the Electrophoresis unit and stained for 20 minutes in LDH activity stain. The LDH activity stain was composed of 0.1 M tris, 1% lactate, 0.05% NAD+, 0.005% nitro-blue tetrazolium, and 0.0005% phenazine methosulfate.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was run to analyze the purity of each LDH purification intermediate. The samples were thawed, vortexed, and centrifuged for 2 minutes at 13.4 RPM. The Affinity Purified LDH was dissolved in an additional 20 uL. Samples were diluted according to this table:

All samples had an additional 4 uL of 5X sample buffer added. The Purification intermediates were heated for 2 minutes at 95 celsius and then centrifuged at 13.4 RPM for 1 minute.The wells of the SDS-PAGE was cleaned with tris-glycine buffer. The samples, LDH standard, and molecular weight ladder marker were loaded into the gel. The gel ran at 120 V for 45 minutes and 140 V for the last 10 minutes. The gel was stained in 50 mL of Coomassie Blue Stain for 1 day and washed with distilled water for 1 day.

Results
Isolation of Cytosolic Proteins: The first centrifuge of the crude homogenate to remove large organelles and cellular debris yielded 81.7 mL of clarified homogenate. The resulting pellet was very large and easily dislodged. This centrifuge was not run at a high enough RPM to pellet cytosolic proteins so LDH was present in the supernatant. The supernatant was cut 45% with a ammonium precipitation and was spun once again. This 45% cut yielded 88.0 mL of supernatant. The supernatant cut was brought up to 65% and centrifuged. LDH was expected to precipitate under a 65% cut so the pellet was kept and resuspended in phosphate buffer. The final volume of 65% cut protein was 6.9 mL.

Affinity Chromatography purification of 65% cut precipitate:

Fractions 6-19 were found to test positive for LDH activity. These fractions were combined for further purification. The final volume of the Affinity Purified LDH was 305 uL.

Size Exclusion Chromatography of Affinity Purified LDH:

Spot Test Excel for this

LDH Activity Assay

Bradford Assay

Tube Label
MQ Water (uL)
BSA Standard (uL) ug Protein in tube
BSA0
100
0
0
BSA1
99
1
1
BSA2
98
2
2
BSA4
96
4
4
BSA6
94
6
6
BSA8
92
8
8
BSA10
90
10
10

Tube Label
MQ Water (uL)
LDH Sample (uL)
CH2
98
2 of Clarified Homogenate (1:10)
CH10
90
10 of Clarified Homogenate (1:10)
65P2
98
2 of 65% cut pellet (1:50)
65P10
90
10 of 65% cut pellet (1:50)
A2
98
2 of Affinity Purified (1:1250)
A10
90
10 of Affinity Purified (1:1250)
S2
98
2 of Size Exclusion purified (1:50)
S10
90
10 of Size Exclusion purified (1:50)

Subunit Identification

SDS-PAGE

Purification Intermediate
Dilution
Sample (uL)
Water (uL)
Clarified Homogenate
1:5
7.5
8.5
65% cut Pellet
1:10
8.7
7.3
Affinity Purified
1:100
3.4
12.6
Size-Exclusion
1:10
8.9
7.1
LDH Standard
------------
5
11

Clarified Homogenate = 81.7 mL
65% Cut= 6.9 mL
Affinity Chromatography= 13.2 mL
Size Exclusion Chromatography= 1.107 mL

Discussion
The most effective recovery of LDH was the Size Exclusion Chromatography. High Activity unit with low total protein shows high selectivity and high purity.
The most effective purification step was Affinity Chromatography.
First, the amount of total protein removed by the Affinity Chromatography is difficult to quantify because there appears to be an error in the analysis data for 65% cut precipitate. The protein from the previous purification step limits the amount of protein that can be recovered by the subsequent purification step. In this case, the total protein recovered by 65% cut was 159 mg, but Affinity Chromatography recovered 350 mg of protein. Purification steps cannot gain protein unless protein is added during the purification process, which none was. The effectiveness of 65% cut precipitation cannot be determined solely by the difference between the difference between the total protein for Clarified Homogenate and 65% cut. However, the total Activity units approximates the selectivity of each purification step when compared to total protein recovered. The difference between the Activity units shows the amount of desired protein lost, which describes the discrimination between other protein. The higher the difference the less effective the purification step was. The difference between 65% cut and Clarified Homogenate activity is the largest and therefore the least effective. For lack of correct data for the amount of protein recovered by 65% cut and Affinity Chromatography this can be qualitatively seen by analyzing the SDS-PAGE. The more bands removed between the steps the more effective. The 65% cut removed only
one band when compared to Clarified Homogenate. On the other hand, Affinity only had 3 bands that were not LDH. Affinity Chromatography removed more bands than the 65% cut. It can be assumed then that Affinity Chromatography removed the largest amount of unrelated protein. However comparing amount of Activity lost for 65% cut and Affinity Chromatography, it appears that Affinity Chromatography removed the most LDH as well making it a less effective step in recovery. It appears that a majority of LDH was still bound to the resin. The Affinity Chromatography may have high selectivity it is not the most efficient step.