Bio 3465 - Lab 10 - Enzyme assay

Bio 3465 - Lab 10 - Enzyme assay - BIO 3465 Plant...

Info iconThis preview shows pages 1–2. Sign up to view the full content.

View Full Document Right Arrow Icon
1 BIO 3465 – Plant Physiology Laboratory 10 ENZYME ASSAYS: Goa l: Enzymes are major important factors that determine biological activities. It is often necessary to extract enzymes from biological tissues and purify them in order to study the enzyme activity. Biochemists use specific activity of enzymes to determine the level of purity of an enzyme. While we do not have sufficient time to purify enzymes during a regular lab class, today’s lab exercise will help you to understand how to determine the specific activity of an enzyme. INTRODUCTION : Malate dehydrogenase (MDH) is an enzyme in the citric acid cycle that catalyzes the reaction of conversion of L-malic acid (malate) to oxaloacetic acid (oxaloacetate) using nicotinamide adenine dinucleotide (NAD +) as a coenzyme. L-malate + NAD + oxaloacetic acid + NADH + H + MDH is found in all eukaryotic cells as two isozymes, mitochondrial (m-MDH) and cytoplasmic (soluble, s-MDH). Prokaryotes contain only a single isoform. However, there are evidences that plants contain more isoforms, some of which are membrane bound while other forms found in cytoplasm, mitochondria and chloroplasts. This enzyme catalyzes the reaction in both direction. In photosynthesis of C 4 and CAM plants this enzyme is involved in converting oxaloacetic acid to malate. In the citric acid cycle, conversion of malate to oxaloacetic acid catalyzed by MDH. In today’s lab you will isolate crude plant extract from spinach leaves and determine the MDH activity in this extract using a simple enzyme assay. Then you will calculate the specific activity of MDH enzyme. Often, in research you need to purify enzymes from crude protein extracts. While you do not purify this enzyme today, determination of specific activity of an enzyme is important to find out the level of purity of the enzyme at
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 2
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 5

Bio 3465 - Lab 10 - Enzyme assay - BIO 3465 Plant...

This preview shows document pages 1 - 2. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online