An investigation into the conditions needed for the successful working of enzymes.

I am going to carry out a series of investigations on the enzyme catalase.

Catalase

Catalase is a hemoprotein with four subunits of equal size. It catalyzes the following reaction:

Catalase is present in a wide variety of tissues from animals, plants and microorganisms. Mammalian liver contains high concentrates of enzyme. Bovine liver catalase has a molecular weight of 250 000 with four subunits of equal size.

Catalase is found in our blood and cells. When we cut ourselves there is a lot of catalase floating around in both the blood and damaged cells. Hydrogen peroxide is a very good antiseptic which when used on a cut comes into contact with the catalase and turns into water and oxygen. Catalase does this extremely efficiently with up to 200 000 reactions per second. The bubbles you would see in the foam produced are pure oxygen being created by the catalase.

Catalase is also present in potatoes and kidney.
Catalase uses hydrogen peroxide as a substrate as well as a hydrogen acceptor.
Catalase is of interest commercially whenever hydrogen peroxide is used as a germicide. For example, in the food industry for disposing of hydrogen peroxide used in pasteurizing milk prior to cheese making.

Outline of investigation

To carryout this investigation on catalase I will:

Take 2 test tubes, pour hydrogen peroxide of a depth of 2 cm into one and pour water into the other to serve as a control.
Add a small piece of liver to each test tube.
Observe.

*Extra

I am also going to test the pH of the solutions to get an idea of what acidity or alkalinity enzymes work best in.

Enzymes

Enzymes are biological catalysts produced by living cells, which have the power of altering the rate of chemical reactions occurring in the body. They are large complex proteins, each chemical reaction requires it's own particular enzyme.

Enzymes are made inside cells. Once it has been formed it leaves the cell and begins its action. These enzymes are called extracellular enzymes. They include digestive enzymes. Other enzymes never leave the cell. They are called intracellular enzymes. They are there to speed up chemical reactions inside our cells. But not only do they speed up reactions but they also control them. All the time thousands of chemical reactions take place inside our bodies and a particular enzyme controls each reaction. Our enzymes make sure that the right reaction takes place at the right place at the right time.

A substrate is the name given to the substance which the enzyme acts upon and product is the name give to the new substance formed as a result. Here is an example of a reaction which is controlled by an enzyme:

Maltase

(enzyme) ...

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Maltase

(enzyme)

Maltose Glucose

(substrate) (product)

The arrows point in both directions, this is because it is a reversible reaction. Most metabolic reactions can be reversed.

Enzymes have five main properties.

They are always proteins.
They are specific in their reaction.
They can be used over again.
They are destroyed by heat.
They are sensitive to pH.

Enzymes can be extracted from organisms and then used in all sorts of scientific

or industrial processes. Biological washing powder contains proteases which are supposed to dissolve protein stains. Proteases are also used in tenderising meat, skinning fish and removing hair from hides. Cellulases are used for softening vegetables, removing seed coats from serial grain and extracting agar jelly from seaweed. Amylases are used for making syrups, fruit-juices, chocolates and other food products. Enzymes can be very useful to us.

What will make a difference?

I think the four key factors that will make a difference to the way in which the enzymes work are:

Temperature
pH
Enzyme activators
Timing

I have chosen to concentrate on the temperature factor for the rest of the investigation with the additional factor of pH.

Fair testing

To ensure I conduct a fair test I will:

Be careful in the measurements of hydrogen peroxide and water.
Be accurate in the cutting and weighing of the liver so all pieces have the same volume and shape.
Take careful observations and notes.
Make sure equipment is sterilised before use.

All of the above will stay the same throughout each test, the only thing which will differ will be the temperature. I will put one set of test tubes in the fridge, keep one set at room temperature and I will heat the other set. This will at what temperature do enzymes work best.

I will collect a set of three results. I will repeat each test for a second time to make sure that I was accurate the first time. So in total I will have six sets of results. In each set there will be an observational result, a temperature result and a test for pH result. This will give me a range.

Safe testing

I am now going to conduct my experiment I will make I will conduct it safely by:

Hydrogen peroxide is corrosive so I will ensure that it is handled with care and that I wear safety goggles when I handle it.
Every that comes into contact with the hydrogen peroxide will be washed thoroughly after use.
As I am concentrating on temperature I will need to use a Bunsen burner for heating the solutions. To ensure that this is done safely a safety mat will be used, goggles will be worn and someone will be with the Bunsen burner while it is on at all times.
During this experiment raw liver will be handled so hygiene is an important factor, hands must be washed.

Predictions

Temperature

I predict that out of the three tests I will be conducting the enzymes will work best in the set at room temperature. This is because I know enzymes work most efficiently at body temperature (37') and the set at room temperature (about 23') is the closest to this. I predict that there will be no reaction in the control because I know that catalase does not react with water.

I predict that the enzymes will work within a narrow range of acidity or alkalinity because these are the conditions that enzymes work in naturally in the body. I predict that the pH will be the same in both the hydrogen peroxide solution and the control as I know for a fact that the pH of water is 7 and I think these are the conditions in the body where enzymes work most efficiently.

Equipment

For this experiment I will need:

Liver.
Test tubes, 2-6 (depending on whether you will do each experiment for each temperature separately or all together.)
Hydrogen peroxide.
Water.
Tweezers.
Bunsen burner.
Tripod.
Safety mat.
Goggles.
Gauze

Further information.

History of enzymes.

Alcohol fermentation is the oldest known enzyme reaction. It was believed to be a spontaneous reaction until 1857 when the French chemist, Louis Pasteur proved that fermentation only occurs when living cells are present. However the German chemist, Eduard Buchnar discovered that a cell free extract of yeast could cause alcohol fermentation. It was later discovered that the yeast cell produces the enzyme, and the enzyme brings about the fermentation. As early as 1783 the Italian biologist Lazzaro Spallanzani had observed that meat could be digested by gastric juices extracted from hawks. Enzymes were found to be proteins and the American biochemist John .H. Northrop discovered that protein was actually the enzyme and not simply a carrier for another compound.

Medical uses for enzymes.

The medical uses of enzymes are illustrated by research into L - asparaginase, which is thought to effective in treating leukaemia, into dextrinases, which may prevent tooth decay. And into the malfunctions of enzymes that may be linked to such diseases as phenylketonuria, diabetes, and anaemia and other blood disorders.

Enzyme supplements can be taken for two reasons.

To correct a digestive problem such as gas or constipation. These enzymes would be taken with a meal.
To help cure any inflammation associated with injuries and arthritis. These enzymes should be taken between meals.

Types of enzymes.

There are three types of enzymes:

Metabolic enzymes.
Digestive enzymes.
Food enzymes.

Metabolic enzymes catalyze the reactions within the cells. The bodies organs,

tissues, and cells are run by metabolic enzymes. Without them our bodies would not work. Among their jobs are helping to turn phosphorus into bone, attaching iron to our red blood cells, healing wounds, thinking, and making a heart beat.

Digestive enzymes break down foods, allowing their nutrients to be absorbed into the bloodstream and used in body functions. Digestive enzymes ensure that we get the greatest possible nutritional value from foods.

Food enzymes are enzymes supplied to us through the food we eat. Nature has placed them there to aid in our digestion of foods. This way we do not use many of the enzymes inside the body during digestion. In two books written by Dr. Edward Howell he develops the theory that humans are given a limited supply of enzyme energy at birth, and that it is up to us to replenish our supply of enzymes to ensure that their vital jobs get done. Howell believes that the more food enzymes you get, the longer and healthier you live.

Food enzymes are destroyed at temperatures at temperatures above 118. This means that cooked and processed foods contain few, if any enzymes. So it is very important to eat lots of raw fruit and vegetables and these are foods in which enzymes are active.

Another role of enzymes in the body is detoxification - the breaking down of toxic substances so that they are excreted and cannot build up and possibly cause harm. Although this is done by metabolic enzymes research shows that enzymes in the food we eat (although not food enzymes) may help our bodies do this.

Enzymes and genetic engineering.

In genetic engineering scientists use restriction enzymes to isolate a segment of DNA that contains a gene of interest, for example, the gene regulating insulin production. A plasmid extracted from its bacteria and treated in the same way can hybridize with this fragments ends of complementary DNA. The hybrid plasmid is reincorporated into the bacterial cell where it multiplies as part of the cells DNA. A large number of daughter cells can be cultured and their gene products extracted for human use.

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