Biology - Molecules and Cells

 Terms and Concepts 

SECTION 5

CHAPTER 2 - Protein-Based Techniques

Chemical analysis in biology is pretty complicated, owing to the size and complexity of the molecules involved in most of the processes.  Think about the stages of respiration and photosynthesis - for almost every conversion of relatively simple carbohydrates, there are enzymes, coenzymes, and carriers involved - mostly huge, intricate molecules whose activity depends upon subtle differences in sequences and conformation.  And all of these molecules have been assembled in the cells and mixed into the cytoplasmic soup - how can a researcher ever pick out just a single molecule to analyze?

 Separation of Proteins by Electrophoresis

One fairly simple method to sort through a mixture of proteins takes advantage of the differences in size and charges among the molecules.  Electrophoresis uses a support medium of some type - paper, various types of gels - to both float the mixture and support an electric field.  The electric field is engaged for a set amount of time, during which the proteins migrate from the positive end of the field (where the mixtures are placed) to the negative end, depending upon the various charges of the amino acids in each protein (and the interactions with the charged medium).  The proteins have been denatured (unwound) to make them interact better with the medium, as well to shut down the activity of enzymes that could interfere with the process.  A protein which is overall more positively charged will migrate farther toward the negative pole during the process.  But that's not the only factor that affects how far the protein moves - smaller proteins will pass through the medium faster than large ones.  When the process ends and the electric field is turned off, the medium gets stained to make the migrated proteins show up.

Introduction to electrophoresis.

More on electrophoresis (go to 1.15).

More on gels.

Doing it (video).

Image of developed gel.

This process can be used to isolate proteins for further analysis, as well as to find differences in proteins in two near-identical systems, such as closely-related organisms.  It has been used to find differences between normal disease organisms and organisms that have developed drug resistances. If a mutated protein is involved in the resistance, it may be different enough from the normal variety to move to a different place during electrophoresis.

Many sources of instructions.

Electrophoresis can also be used to analyze single molecules, of DNA as well as proteins.  If a batch of a single protein is treated with enzymes that clip the sequence wherever a particular type of peptide bond exists, the resulting bits can be separated by electrophoresis.  Each bit can be analyzed in a similar way, and rough sequences can be built, as well as isolating critical differences between very similar proteins.

Protein analysis techniques (pdf).

A high-tech variation on this basic idea is mass spectroscopy, which uses dispersal of materials as gases and movement through varied-length magnetic fields (the longer the track, the more separation between similar-but-different molecules can occur).  This can be used to separate and identify a wide variety of organic molecules, and can be sensitive to tiny differences.  The technique was originally called atomic mass spectroscopy and was used to distinguish isotopes from one another.  A very good ( = expensive) mass spectrometer can identify a particular molecule, different by just an atom or even a neutron, by how long it takes to get from the intake to the meter.

Introduction to mass spectroscopy / spectrometry.

How it works.

Mass spec readout.

 This takes advantage of the specificity of antibody molecules produced by mammals.  Horses or mice are used.  Antibodies are specifically shaped to connect to antigens, molecules foreign to an organism.  The animals are exposed to particular antigens, then they have spleen cells removed, isolated, and fused with a type of cultured cancer cell that will allow them to replicate indefinitely.  Early cultures are tested to see which ones are making the appropriate antibody, and the ones that are cultured extensively (in the lab, or inside a mouse if lab culturing won't work) enough to harvest usable amounts of released antibodies, which will react when exposed to that particular antigen (or at least a part of the antigen molecule, called an epitope).  Because the process clones just one type of cell, it is said to produce monoclonal antibodies (mAb).

Procedures for producing monoclonal antibodies (large manual pdf).

Use of epitopes.

More on monoclonal antibodies.

Where in a cell are particular proteins active?  That is a question that can be addressed with green fluorescent proteins derived from species of jellyfish that glow.  The approach involves altering the genetics of a zygote:  the gene for the fluorescent protein is "tagged," inserted into the very end of the gene for the target protein.  When the target protein is made and moved, it has the fluorescent protein attached to it.  Then, cells can be placed under a special microscope that makes the fluorescent proteins glow.

Basics of tagging.

The microscope.

Image gallery.

Modern drug discovery is only slightly based on looking for biologically-active compounds from other organisms.  That can be a starting point, but now, computers can alter small parts of molecules in virtual space and make predictions on how they will interact with known biological materials.

An introduction to targeted drug discovery (pdf).

Virtual chemistry is not "real," so follow-up testing needs to be done on promising compounds.  But before anything happens, the compound must be patented, protected as a unique invention.  The patent protects the developing company, and there is no sense testing a compound you wont have the sole right to use.  In the U.S., drug patents last 16 years, after which other companies (usually with no testing facilities) can then produce and sell generic versions of the compounds.  A compound might take 12 years to reach approval for sale (and the vast majority of them never prove usable / marketable), so new drugs tend to be very expensive between release and patent lapse.  Companies are trying to underwrite their research and development costs on both successful and unsuccessful compounds (plus make sometimes huge profits).

More on patents and generic drugs.

Generic drugs - what are the requirements?

A pay-to-view site that just tracks patents (entrance portal).

Actual activity of these new compounds must be tested.  If cell cultures are available, that may be the first step.  A culture would be cells (generally human) that are kept alive in dishes.  There are some cancer cultures and a few other specific disease cultures, but they are just groups of cells in dishes.  Some very important information can be derived from culture tests, but cultures have two major limitations:  they are isolated, there are no influences like what would exist in the body;  it's unclear how much the cells have changed on adapting to a cultured existence.  They may be very different from the cells they started as.  However, researchers can assess some basic cellular responses from cultures.

Information on cancer drug screening for a type of cancer(pdf).

Basics of cell cultures.

The next step in testing involves animal models.  These models require some features:  they need to be comparable to humans and they need to be able to sustain whatever condition the drugs are targeted against.  It helps also if they are small, hardy, cheap to keep, easy to handle, and have relatively short lifetimes and a high reproduction rate.  Mice are very often the model of choice, so much so that many strains of mice have been developed that support conditions that mice typically would not get.  There are, of course, major differences between models and humans, so that there are many instances of drugs that worked in models but not in humans, or which produced dramatic side effects that didnt appear in the models.  Currently, the U.S. Food and Drug Administration (FDA) requires early testing on two different animal models to try to address those issues.

Mice "adjusted" to be prone to certain cancers.

Some problems with animal models.

Making the test animals comfortable-?

Human testing of chemicals involves clinical trials in several phases, all supervised (or at least the results are reviewed) by the FDA.

Introduction to clinical trials.

Phase 1 Clinical Trials are used to see if its even feasible to expose humans to the chemical.  A small group of healthy humans are given the drug and checked for major side effects while researchers try to follow how a human metabolism deals with the chemical.  This is basically to assess the safety of the chemical, and to see if humans can easily tolerate exposure to it.

Phase 1.

In Phase 2 Clinical Trials, a small but statistically-significant group (if such is available - some conditions just aren't common enough to provide good numbers) of patients with the target condition are given the drug.  Blinding varies in this stage.  Now actual curative results are being sought, as well as side effects that might have not shown up before.  This will be a double-blind test, or possibly triple-blind.  To proceed to the next step, the FDA will review not just safety and efficacy, but efficacy relative to drugs that are already on the market:  to move forward, your drug should outperform currently-available treatments.

More on the steps.

More.

In Phase 3 Clinical Trials, a much larger group (if possible) is tested.  This group should be double (sometimes triple) blind, if numbers allow.  This will double-check effects, both primary and side effects (and rarer side effects should show up in a large group), and sometimes will reveal issues with administering the drugs and following up with patients.  If this all goes well, the drug may be approved for sale.

Problems with Phase 3 (pdf).

More on clinical trials.

Phase 4 Trials are usually an indication that something might not be right with the released drug.  It is required for some pediatric applications but can be very disorganized in other instances, relying on optional reporting. This is an attempt to gather information on how the drug is affecting the broader population now exposed to it.  Sometimes a very rare (but very serious) side effect only appears at this stage.  It make take years for anecdotal reporting to suggest a problem, but it does sometimes happen.

Some companies see Phase 4 as an opportunity.

Terms and Concepts

In the order they were covered.

Electrophoresis of Proteins  
Mass Spectroscopy   
Monoclonal Antibodies 
Epitope
Fluorescence Tagging 
Drug Development
Drug Patents
Generic Drugs
Cell Cultures (for testing)
Animal Models
Mice Strains
Clinical Trials