Chemistry

The most abundant biological macromolecules in all cells and parts of cells are a group of compounds called proteins. There are different varieties of proteins ranging from relatively small peptides to large polymerized molecules with huge molecular weights in thousands and millions. Proteins have diverse biological functions, especially in the transfer of genetic information. The base unit of a protein molecule is an amino acid. All proteins, whether from the most ancient lines of bacteria or from the most complex form of life, are constructed from the same ubiquitous set of 20 amino acids, covalently linked in characteristic linear sequences (Nelson  Cox, 2004). These amino acids serve as the alphabets in which the structure of protein is written. Therefore, proteins can be said to be polymers of amino acids.

Proteins can be hydrolyzed (that is, broken down) to the various amino acid residues by several methods. Amino acids also, can be classified into different groups or classes. According to Nelson  Cox (2004), they can be grouped into nonpolar, aliphatic R groups aromatic R groups polar, uncharged R groups positively charged R groups and negatively charged R groups. Proteins also have three-dimensional structures which characterize and reflect the function of each. These structures are stabilized by numerous weak interactions, especially hydrophobic interactions, hydrogen bonds, and ionic interactions. Proteins also have secondary structures which refer to the local conformation of some part of a polypeptide. Apart form the secondary structure, proteins also possess tertiary and quaternary structures (Murray, Granner, Mayes,  Rodwell, 2006). Tertiary structure refers to a proteins overall 3-D arrangements of all the atoms in space, while the arrangement of identical or different protein subunits constitute the quaternary structure.

Enzymes are the most significant and specialized proteins. They have extremely high catalytic powers, high degree of specificity for substrates, accelerate chemical reactions tremendously, and they function in aqueous solutions under very mild conditions of temperature and pH (Nelson  Cox, 2004). Almost all enzymes are proteins except some RNA enzymes such as a group of RNA molecules. Enzymes do not form a chemical bond with the substrate and after the reaction, the products are released and the enzyme returns to its normal shape. (CITATION) Enzymes function just like catalysts. If a catalyst is absent, the energy required to cause a chemical reaction is quite large and the speed of the reaction extremely slow, but the presence of a catalyst ensures that the energy requirement is lowered and that the reaction takes place faster (Heriot-Watt University 2001). Likewise an enzyme which is biological. It reduces the activation energy by forming an enzyme-substrate complex which increases the rate of reaction. The rate of an enzyme-catalyzed reaction may be measured by (1) the disappearance of substrate or (2) the appearance of product (Enzyme Function Effects of Substrate Concentration on Alkaline Phosphatase Activity, 2010).

Alkaline Phosphatases are a group of enzymes found primarily the liver (isoenzyme ALP-1) and bone (isoenzyme ALP-2) (Kaslow, 2010).Alkaline phosphatase is an enzyme involved in catalysis of the cleavage of esters of phosphoric acid (Enzyme Kinetic Analysis of Alkaline Phosphatase, 2010). This enzyme is found in relatively large quantities in the human and animal tissues, especially in the liver, kidneys, blood, intestine, bones, and the breast.

This experiment attempts to verify the hypothesis that increasing enzyme concentration will increase the rate of a chemical reaction. In order to test this, a synthetic substrate (p-nitrophenyl  phosphate  (pNPP)) was used. Alkaline phosphatase (ALP) catalyses the hydrolysis of p-nitrophenylphosphate at pH 10.4, liberating p-nitrophenol and phosphate, according
to the following reaction (Atlas Medical, 2005)
pNPP (colourless)   Pi  pNP (yellow)

Materials and methods
The materials used for this experiment includes six test tubes, water bath, 0.1M NaOH, well plate, and a plate reader.

Six tubes were labeled with the numbers 1, 2, 3, 4, and 5. The sixth tube was labeled blank. All the tubes were pre-incubated in a water bath for 5 minutes at 37oC. After pre-incubating the tubes for five minutes, different volumes of enzyme extract were added to each of the tubes at 30 second intervals. The volumes used were 0.1ml, 0.25ml, 0.5ml, 0.75ml, 1.0ml, and 0.0ml for tubes 1, 2, 3, 4, 5, and 6 respectively. Incubation proceeded for 10 minutes. The reaction was stopped in each tube by adding 1ml of 0.1M NaOH serially at 30 second intervals. The contents in each tube were again mixed together. Finally, 200L of the contents of each tube were pipette across one row of a well plate and the optical densities read with a plate reader.

Results
Table 1. Data for the experiment.
Tube number123456 (blank)ml of extract0.10.250.50.751.00.0OD at 400nm0.1620.2190.3160.4320.5250.117OD (400 nm) - blank0.0450.1020.1990.3150.408mole10 mintube0.01170.026520.051740.08190.10608molehtube0.07020.159120.310440.49140.63648molehml of extract0.7020.636480.620880.65520.63648

Calculations
The conversion factor used for calculating the amount of p-nitrophenyl in each tube was
Absorbance of 1.0  0.26mole p-nitrophenyl
For the first tube, since the recorder OD (optical density) at 400nm is 0.162, subtracting it from the OD of the blank tube will give 0.045.
Conversion of OD10 minutes to mole10ml in tube 1 will be 0.045 X 0.26  0.0117
Converting it from per 10 minutes to per hour will be (0.011710 mins) X 60 mins  0.0702
To convert to per ml of extract, 0.0702 will be divided by the initial amount of enzyme extract (0.1ml), resulting in 0.702.

Figure 1. Graph showing the relationship between mole10 mintube on the y-axis, and enzyme extract (ml) on the x-axis.

Discussion
The results of the above reaction have validated the hypothesis that increasing the enzyme concentration increases the rate of reaction and therefore the product yield. This is better illustrated by the graph in Fig. 1. The graph is almost a straight line linear graph with a direct proportionality between the product yield (mole10 mintube) and the amount of enzyme extract in ml. Increasing the enzyme concentration increases the volume of p-nitrophenyl that was produced. Table 1 shows the increase in the amount of products.

The chemical basis of this is that increasing the enzyme concentration increases the amount of enzyme available for the reaction. Enzymes possess pockets which are called active sites. When enzymes act upon chemical reactions, they open their active sites in order to bind to substrates. Usually, the active site encloses a substrate, sequestering it completely from solution (Nelson  Cox, 2004). There is also the formation of an enzyme-substrate complex. When enzyme concentration is low, there are low amounts of active sites, causing competition among the substrates for the few active sites present in the solution. As the enzyme concentration increases, the numbers of active sites correspondingly increase, so that more substrates are being bound per minute, ultimately resulting in an increased reaction rate.

For a given enzyme concentration, the rate of reaction increases with increasing substrate concentration up to a point, above which any further increase in substrate concentration produces no significant change in reaction rate at a certain point, the rate of reaction levels out (Enzymes, 2010). This means that increasing enzyme concentration will no longer have any effect on the rate of reaction. As the amount of enzyme is increased, the rate of reaction increases, but if there are more enzyme molecules than are needed, adding additional enzyme will not increase the rate (Enzymes).

Figure 2. Enzyme activity against enzyme concentration. Source Manual of Clinical Enzyme Measurements (1972).

The last tube (tube 6) serves as an experimental control. This confirms the fact that when enzyme is not added, no reaction occurs.

This experiment was successful in that it has been able to evaluate the effect of increasing enzyme concentration on enzyme activity. It has held constant all other factors that can affect the rate of a chemical reaction such as temperature, pH, substrate concentration, reaction time, etc. In biological systems, this experiment will aid in understanding kinetic analysis of enzymes. Enzyme concentration is usually the most significant difference between routine in vitro assays and in vivo conditions, as it is well known that many intracellular enzymes are present in vivo at much higher concentrations than used in vitro (Aragorn  Sols, 1991).