Audience: Teachers and Students

Faculty: Dr Stephen Graber Ph.D.

OUTLINE

Summary

Proteins carry out most of the tasks necessary for the survival of living organisms. Creatine Kinase (CK), also known as phosphocreatine kinase (CPK), is a type of protein called an enzyme. Enzymes catalyze or encourage biochemical reactions. The main function of CK is to add a high-energy phosphate group to creatine to make phosphocreatine. Phosphocreatine can be used as a quick source of energy by our cells. Most of the creatine kinase in our bodies is found in muscle cells. When muscle cells are damaged by disease or injury they often rupture which allows the creatine kinase to leak into the bloodstream. Normally very little creatine kinase is found in the blood, so a rise in the amount of creatine kinase in the blood indicates that muscle damage has occurred, or is occurring. This measurement is known as a Creatine Kinase Test and can be used to diagnose heart attacks, muscle disease and strokes. In this inquiry experience you will see how creatine kinase activity can be measured and used to purify creatine kinase from a complex mixture of cellular proteins so that its properties and functions may be studied.

Objectives:

This inquiry experience will help you:

• Become familiar with some of the equipment and procedures used to purify proteins.
• Understand how the activity of a protein may be measured and quantified.
• Use a computer program to analyze and graph scientific data.
• Understand how muscle cells acquire the energy to do work.

The week’s activities will be divided into blocks which are described below. For each of the blocks you will be provided detailed written descriptions and several people familiar with the procedures will be present while you perform them.

Block I- Introduction

In the first block you will have a chance to become familiar with the lab and equipment you will use in each of your experiments.

  

Block II- Protein Purification

 

In the second block you will use Ion Exchange Chromatography to partially purify Creatine Kinase from an extract of cells. Ions contain small amounts of electric charge and Ion Exchange Chromatography separates proteins based on differences in the amount of their ionic charge. The proteins in the mixture will be separated into groups (called “fractions”) with low, medium and high ionic charge. You’ll save these fractions for use in the next several blocks.

Block III- SDS Polyacrylamide Gel Electrophoresis

In the third block you will get to see all the proteins you separated in Block II. You will use a technique called SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) to separate the proteins in your fractions based on their size (molecular weights to be precise). After staining the gels overnight you’ll see the individual proteins in each fraction as narrow bands arranged vertically by size. By comparison with a sample of previously purified creatine kinase you ought to be able to make a good guess as to which fraction contains the creatine kinase.

Block IV – Measuring Creatine Kinase Activity

In the fourth block you will measure the creatine kinase activity in each of the fractions prepared in Block II and compare them with a creatine kinase standard of known activity. You will use a spectrophotometer to measure the amount of a colored product produced in the biochemical reaction catalyzed by creatine kinase. You will also finish staining the SDS-PAGE gel from Block III and should be able to identify the protein band responsible for the enzyme activity.

Block V – Measuring Total Protein Content

In the fifth block you’ll measure the total amount of protein in each of the fractions from Block II and in the starting material. You’ll use a spectrophotometer to measure the amount of a colored complex that forms in the presence of protein. The amount of protein in your samples is determined by comparing the color that forms in your fractions with the color that forms in the presence of known amounts of a protein standard. These measurements will allow you determine how much purification was achieved by the ion exchange chromatography and help you understand how enzyme activities are usually expressed.

Block VI – Computer Assisted Data Analysis and Scientific Graphing

In the last block you’ll use a computer to analyze the data collected in the enzyme and protein assays. You’ll see how a technique called “linear-regression” can be used to precisely determine the activity and protein levels in your samples. This technique relies on simple algebra and is the basis for many of the diagnostic tests run by doctors and hospitals. You’ll also use the computer program to prepare graphs of your standard curves which you can use in your web authoring presentations if you wish.

Developed on July 11, 2005 by Sohail Khan