Polyphenoloxidase Enzymatic Reactions of Kinetical Energy


X. Inhibition of potato enzymes by Experimentation
Jerry The Great
Laboratoire de Science, Institut de la Recherche Agronomique/Universite de Murray State
UA327, bv 1369, 42071 Murray, Kentucky, USA

INTRODUCTION

Polyphenoloxidase (E!.10.3.1), an enzyme not uncommon in that of a potato, or any other plant for that matter, has been found to play an integral role in that of plant life. A recent lab experiment performed by four notable scientest wanna be's, Jeffy Belms, Trisha Bean, Rachel Biakabatuka, and myself turned up very fascinating, of sorts, results which could possibly shake the very foundations of the earth-time continuum of the modern botanical world.

Before jumping head first into the kinetics of phenoloxidases, very little was known about the aforementioned enzyme and excitement ran voraciously through our veins as we prepared to study the majestic enzyme for visions of fame and glory pounced in and out of our heads as we arranged for all the substances which we would use to be put in place.

Later, through a series of difficult procedures which we had conjectured through countless hours and reseach and debate and the reading of a lab manual, we finally ambled across what we believed to be the answer to all botany related questions dealing with fruit and why the "fruity" part of the plant turned a brownish color when exposed to the gas oxygen, which is what we found to be the exact cause of that reaction. This enzyme is also involved, thanks to nature, in its defense of herbivorial predators with its productorial tendencies. Even more fascinating, as we delved even further into the project, was how phenoloxidase is indirectly responsible in the plants healing process. The dark brown products formed as the result of phenoloxidase action also act to seal off bruises and cuts so as the protect the internal cells of plants.

Although I initially believed the investigation of the phenoloxidase enzyme to monitor the enzyme activity, and while it did pertain to my thesis, I felt it to deal more with determining the effect of enzyme and substrate concentrations on enzyme activity, which particularly fascinated me. However, during the processing of the enzyme, I came up with a goal of my own - to study the isolation and characterization of the polyphenoloxidase enzyme in order to gain more precious information about its enzymatic and structural properties to a further degree, which led me to hypothesize on the overall importance of the experiment, to understand and see how enzymes perform their pre-meditated duty.



MATERIALS AND METHODS

Preperation

All necessary steps and provisions were taken with an insane amount of care, which called for someone to blend a potato, strain the solution, and pour it into the smallest containers possible so as to minimize potato enzyme contact with the air. If these extreme precautions would not have been taken, the question of our sanity might have come into play sooner or later during the bounds of this experiment. After this had all been accomplished, it was necessary to place the potato extract in a container of ice water, thereby "freezing" the race against the enemy, time. (Of course, I just naturally assume all this was done quickly so as not to compromise the accuracy of our experiment).

After the potato had been put through the process of homogenization, which is essentially the combining or dissolving of substances, we found it our customary obligation to indulge in the pouring of 30mL of phosphate buffer and 15mL of catechol into 5 Klett tubes which had been provided for us, used in obviously equal proportions so as not to disrupt any fluidity in our experiment that we would hopefully encounter so that the experiment could move along at a fairly rapid pace, considering the length of time it would take to conclude the experimentation.

Plotting the Data


As is typical of science, after the four of us reached a general degree of agreement, we were forced to contemplate yet another problem dealing with the confusing potato enzyme, phenoloxidase! As abruptly as we had finished the first step in our experiment, another was shoved right back into our faces, but thankfully I began to feel very scientific. Questions about the rate of enzymatic reactions and how they change when the concentration of enzyme is increased or decreased popped into my head with increased buoyancy (it was only later that I found out this was a mandatory assignment in the lab) and I began to ponder the initial rate of each run from a straight line drawn from the first couple of readings. After tabulating the rates for corresponding enzyme concentrations, which I incidentally came about when I mixed the phenoloxidase potato enzyme, phosphate buffer, and catechol into a container and measured the reaction in a machine oddly known as the "Spec-20," or colormeter, I lived to, ta da, plot a graph!

Let it be deuly noted that all graphs were formulated after several exhausting data taking moments in the Botany Lab.

RESULTS AND DICSUSSION

Results


After the successful homogenization of the phosphate buffer, catechol, and phenoloxidase substances and its placing in the Spec-20, I became aware of a certain pattern which seemed to protrude from all the readings that I couldn't help but contemplate about. To quicken the reaction of the enzyme and catechol/buffer substance, the small tube like container was shaken, but not stirred, before its quick meeting with Spec-20, which gave me the following data.




with regret, all graphs and tables are not available (but they were absolutely beautiful)

Discussion I


After I had finished dictating the experiment for our group, I just couldn't help but notice a little oddness which seemed to "permeate" from the page my partners had just taken down, particularly in the 1-2mL of enzyme zone after about 1-2 minutes of experimenting. Questions of why this happened popped into my head, bringing wild conclusions of mass hysteria within the confines of the little test tube which contained the product of phosphate buffer, catechol, and phenoloxidase enzyme. When the ultimate pondering of such data and the wrongful note taking of such data was flung into my head, I decided that maybe I should graph or plot my data and compare it to others, to perhaps solve the mysterious case of the buffer/catechol/enzyme reaction.

Results II

Suddenly, the immense amount of pride and joy I had achieved from successfully graphing the figure was all but destroyed as new questions of what would happen when I increased or decreased the substrate concentration. Confound it all!

Dashing over to the excess supply of phosphate buffer and enzyme concentration, I grabbed as much as my arms would hold and ran back to my table and began to work again at a furious pace. I was aware that I had to use the same amounts of phosphate buffer and enzyme concentration, but the volume, I had to change the volume of catechol and distilled water or all would be lost! Would the effect of substrate concentration actually change the enzyme activity to a noticeably different level was a key contemplation of mine and essential to the task at hand.

Again it was necessary to mix the enzyme/catechol/buffer into a relatively small mixture, but the substrate concentration being boosted up to a larger level than before. An initial reading was taken immediately in the Spec-20 and every thirty seconds which departed the grasp of planet Earth would be taken for the next seven and a half minutes so as to appease the botany instructors.

Discussion II

Once again, I couldn't help but be aware of the presence of what seemed a noticeable peculiarity, such as the obvious neglect of conformity on the behalf of the substrate. Why it did this, I had to formulate on the fact that was presented to me by the Spec-20. For some reason it just seemed slightly odd that the amount of substrate in the beginning of the experiment actually made no positive contribution to the procedure. After all, it seemed that it inhibited, if anything, the reaction that we were studying, which was my initial and genuine hypothesis. Later, however, the reaction began to occur at the rate which I had come to know it, particularly fast at first and then generally slowing down to a point (although sometimes it would take longer than other times) where little reaction could be detected.

I Could Have Been a Contender! 1