EFFECTS OF VARYING TEMPERATURES, PH LEVELS, AND MALATE

DEHYDROGENASE CONCENTRATION ON ENZYME ACTIVITY

Jake Oshlo
Biology 160
3/30/10
INTRODUCTION
All processes in the world operate on the principles of chemistry and chemical reactions.

Whether we consciously acknowledge it or not, intermolecular interactions are taking place

throughout all aspects of the world. Chemical reactions are constantly taking place as bonds

between atoms break and form to shape the world as we see it. Living organisms, in particular,

are a hotspot for chemical reactions. In a human body, billions upon billions of chemical

reactions take place every second. These reactions are a necessity to life and other natural

processes of the world.

All living organisms, from a leaf on an ash tree to an elephant roaming the savannahs of

Africa, are comprised of billions or even trillions of cells. Cells are the foundation for any form

of life and cellular function provides the means for life to exist. Each cell contains chemicals

that are constantly moving and reacting to maintain the life and function of not only the cell, but

the entire organism that it is a part of. If the chemical reactions within cells ceased, the cell

would die. If such a stoppage occurred on in many cells, the organism would not survive. The

reactions are essential to the persistence of life. Most cellular reactions, though, cannot maintain

life unaided. They would normally take place at too slow of a rate to be effective at carrying out

cellular processes. Enzyme catalysts serve to counter this lack of time-efficiency. The enzyme

catalysts accelerate the reactions within the cell. Without these enzymes, the reactions would

occur too slowly and the cell would lose its ability to function at a pace necessary to produce and

live.
 Chemical reactions require a certain amount of energy to proceed. This amount is

referred to as the activation energy. Within cells, most reactions require an activation energy

that is difficult to obtain. This results in the reactions occurring at a very low rate due to very few

molecules having sufficient energy. Enzyme catalysts are proteins that lower the activation

energy by binding to molecules and arranging them in a manner that favors reaction between

molecules. The molecules bind to a specific cluster of amino acids on the enzyme that forms a

groove. This groove is called the active site. The active sites of different enzymes are very

specific for certain substrates.

There are a number of variables that enzyme function is sensitive to. They aid the

reactions at different rates depending on the pH level, the temperature, and the concentration of

the enzyme. The effect of each of these variables on the activity of the enzyme MDH was

examined in the experiment as it was used to oxidize NADH to NADH+.

MATERIALS AND METHODS

The procedure is from Duncan et al Cell Biology Lab Laboratory Manual 2010. A

cuvette was initially prepared to blank the spectrophotometer at a wavelength of 340 nm. 1 mL

of phosphate buffer (pH 7.5), 10 µL of oxaloacetic acid, and 10 µL of NADH were added to the

cuvette for the blank. 10 µL of the 1X enzyme solution were then added to the cuvette and it was

mixed before being placed in the spectrophotometer. Absorbance values were taken every ten

seconds for two minutes in order to analyze the decreasing presence of NADH as it was oxidized

to NADH+. This process was repeated two more times, with the concentration of MDH being the

only changing variable. The amounts of phosphate buffer, oxaloacetic acid, and NADH put in

the cuvette remained constant, while instead of a 1X solution, a .5X and finally a .25 X enzyme

solution were added. After collecting the absorbance data on the individual solutions with each

of the three enzyme concentrations, plots were made of the absorbance readings at 340 nm as the
reactions proceeded over a period of two minutes. This data was then used to calculate the units

of enzyme activity for each of the three enzyme concentrations.

For the next set of data, the effect of pH on the effectiveness of MDH was examined. The

pH of the buffer in the original assay was 7.5, with which data had already been obtained. The

assay was repeated with the same amounts of NADH, buffer solution, and oxaloacetic acid, and

the same amount and concentration of enzyme solution. The buffer solution in the cuvette had a

pH of 4.5, though, to test the effect of lowering the pH. The assay was performed again with a

buffer solution of pH 10 to test the effect of raising the pH. After collecting data on all three pH

levels of the buffer solution in the total solution, plots were made of the absorbance readings to

compare the effects of varying pH. With this data, the units of activity were calculated for the

three different pH levels of the buffer solution.

The effect of temperature on the activity of MDH was examined next. The original assay

was performed at room temperature so the next assays were taken at varying temperature. A

solution of 1 mL of phosphate buffer (pH 7.5), 10 µL of oxaloacetic acid, and 10 µL of NADH

was set in an ice bath to lower its temperature to 1º Celsius. The 1X concentration of enzyme

solution was then added to it, mixed, and placed in the spectrophotometer to gather the necessary

absorbance data. This process was repeated with the solution being warmed to 37º prior to

having the MDH mixed into it and placed in the spectrophotometer. The assay was repeated once

more with the addition of MDH enzyme solution that had previously been boiled before being

analyze in the spectrophotometer. The data gathered was plotted to observe the effect of

temperature on the enzyme activity, then used to calculate the units of activity for each

temperature.

RESULTS
 Lowering the concentration of the enzyme solution had a clear effect on the enzyme

activity in oxidizing NADH. The absorption per minute of the solution decreased at a much

faster rate in the 1X enzyme solution than in the .25X and .5X concentration (See table 1). The

enzyme activity at .5X concentration was significantly lower than at 1X concentration. The units

of activity at .25X concentration of enzyme solution were slightly higher than at .5X, but still

much lower than at 1X concentration. (See figure 1) Altering the pH of the solution from the

optimum 7.5 changed the absorption per minute and enzyme activity as well. The absorption per

minute of the solutions with pH of 4 and 10 decreased at a slower rate than with a pH of 7, and

the pH of 10 decreased faster than the pH of 4 (See table 1) The enzyme activity followed the

same pattern in relation to the absorption per minute, with solutions of pH 4 and 10 having less

activity than with a pH of 7.5, and the pH of 10 being greater than the pH of 4. (See figure 2)

The effect of temperature on enzyme activity was also noticeable. When the MDH solution was

boiled, it resulted in no enzyme activity. When cooled to 1º Celsius, the activity was greatly

reduced from at room temperature. Warming the solution to 37º Celsius also reduced the enzyme

activity, more so than cooling it to 1º Celsius. (See figure 3)

DISCUSSION

The results of the experiment went as expected for the most part. When comparing the

different MDH enzyme concentrations, it is logical to assume the higher concentration of

enzyme would produce the most enzyme activity; more of the enzyme would help the reaction

occur faster. The experiment showed this to be true; the 1X concentration had higher activity

than .25X and .5X. In that sense, it would be expected that the .25X concentration of enzyme

solution would have less activity than the .5X, but it was slightly more. (See figure 1) This could

be a result of poor technique, measuring, or an error in reading the absorption values. In the three
levels of pH, it is expected that the ideal pH for MDH, 7.5, would produce the most activity.

Enzymes denature at high and low levels of pH. This held to be true in the experiment. The

activity of the solution with a buffer of pH 7.5 was more than the solutions with buffers of pH 4

and 10. In the tests of the effects of the different temperatures of the solutions, there were some

discrepancies between the results and what was expected. As was expected, the enzyme activity

decreased significantly when cooled to 1º Celsius. The cooling takes away energy needed for

reactions. When the enzyme solution was boiled to 100º Celsius, there was no enzyme activity at

all. This is a reasonable result because at high temperatures, the enzymes denature and can no

longer act as catalysts in reactions. However, when the solution was raised to 37º Celsius, it

would be expected that the enzyme activity would increase from that of a solution at room

temperature. In the experiment, though, the activity decreased greatly. This could have been due

to a problem with the enzyme solution or an error in the experiment.

Studying the effects of enzyme catalysts on reactions is an invaluable tool that can be

used in a number of useful applications. We can better understand how body functions and works

and learn more about its inner workings. Knowing this can help make for a better lifestyle.

Knowing how to maximize the efficiency of enzymes and chemical reactions, we can better

prepare and apply different medical technologies that can be perfected to improve and save lives.
 Abs/min(slope) U (enzyme activity)
Concentration
0.25 X -0..312 0.05
0.5 X -0.192 0.03
1X -0.996 0.16
pH
4 -0.552 0.09
7.5 -0.996 0.16
10 -0.834 0.13
Temperature (oC)
0 -0.246 0.04
25 -0.996 0.16
37 -0.012 .02
100 0.0 0.0