Molecular Biology and Ldh

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  • Topic: Lactate dehydrogenase, Molecular biology, Enzyme assay
  • Pages : 21 (6183 words )
  • Download(s) : 162
  • Published : January 14, 2013
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*Running title: Isolation and Characterization of Avian Lactate Dehydrogenase

To whom correspondence should be addressed Sylvia DaoudKinze and James Proestes, Department of Biochemistry, Portland State University Professor, Portland Oregon, 97207-0751; E-mail: sylviakinzie@gmail.com and proestoj@gmail.com

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Keywords: Lactate, Dehydrogenase, Avian, Bradford Assay, Affinity Column

Background: Lactate Dehydrogenase also known as LDH is an important NADH dependent enzyme in metabolism that catalyzes the conversion of Pyruvate to Lactate. Results: Catalytic activity was detected from chicken breast muscle Conclusion: Copious amount of catalytic activity was detected, indicating that purifications and extraction of chicken tissue is done correctly. Significance: Learning and characterizing avian lactate dehyrogenase activity, and learning proper lab protocols in a biochemical setting.

Summary
Isolation and Characterization of Avian Lactate Dehydrogenase experiment consist of extraction of LDH from chicken breast muscle, then purifying the LDH obtain from chicken breast through affinity column chromatograph, determine protein concentration by Bradford assay and then determining the amount of catalytic activity through activity assays. Through Bradford assays, copious amount of protein concentration was detected, thus showing purification methods with affinity was successful, and with LDH activity assay, results concluded that there was catalytic activity among the protein concentration. The isozyme was then determined and separated by Agarose gel electrophoresis. The molecular weight of the purified protein was then obtain through SDS-Page method. Westren blotting method was induced, but failed due to the wrong reagents were ordered.

Introduction
Lactate Dehydrogenase catalyzes the conversion of pyruvate to lactate. There are four different distinct classes of LDH enzymes. Two of the classes are cytochrome c- dependent enzyme, each acting on either L-Lactate or D-Lactate . The other two classes are NAD(P)-dependent enzyme that also either acts on L or D- Lactate. In muscles tissue, LDH is crucial to maintaining levels of NADH and NAD+, especially under anaerobic condition. In Glycolysis, which is a metabolic pathway that converts glucose molecule into pyruvate and in the absence of oxygen, the only source of ATP comes from Glycolysis, although this metabolic pathway consume copious amount of NAD+ to fuel this process. Lactate Dehydrogenase serves to replenish the depleted NAD+ so that the glycolysis could keep producing ATP. LDH is applied in various medical usages as a marker. Hemolysis, which is the breakdown of blood cells could be marked by LDH by anazlyzing the blood sample to see if there is an increase in LDH concentration. LDH could also be a marker for myocardial infarction. In pathology, high levels of lacatate dehydrogenase in cerebrospinal fluid are an indication that the patient has bacterial meningitis. In many cases of viral meningitis, high LDH, in general, indicates the presence of encephalitis and poor prognosis. In HIV patient, LDH is often measured as a non-specific marker for pneumonia due to pneumocystis jiroveci (PCP). The objective of this experiment is to extract LDH from chicken breast tissue and purifying the LDH. Purification can be achieved by homogenization, ammonium sulfate fractionalization and affinity column chromatography. After purification have been achieved, determination if protein composition exist will be determined through Bradford assay. To see if catalytic activity is present, LDH activity assays will be analyzed. Then Agarose electrophoresis method was used to separate the isozymes of LDH. To acquire the molecular weight of the purified protein, SDS-Page method was used.

Methods

Extraction of LDH from chicken breast muscle
CFE Preperation
Cell Free Extract (CFE) of chicken breast was prepared through...
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