Factors Affecting Enzyme Rate of Reaction - Assignment Example

Experiment to Understand Factors Affecting Enzyme rate of Reaction Introduction: Enzymes like all chemicalsare affected by environmental conditions. Enzymatic reactions are affected by several factors such as temperature, pH and concentration. In this experiment we shall be studying the effects of temperature, Ph and substrate concentration upon the catalytic activity of an enzyme- alkaline phosphatase. In the human body, this enzyme catalyzes the removal of phosphate group from the DNA. However for our purpose we shall be using a colourless substrate-paranitrophenol (PNPP). The enzyme, alkaline phosphates cleaves the colourless substrate to give nitrophenyl and phosphate. Nitrophenyl being a colourful product maybe measured spectrophotometically. Requirements: PNPP ( substrate) Enzyme 10 cuvettes Buffer (NaoH-glycine) Pipettes (10 nos) Water bath Ice bath Thermometer De-ionized water Basic Experimental protocol: a. We need to make control and experimental cuvettes. Label the experimental ones- 1E,2E,3E,4E and 5E and label the control ones 1C,2C,3C,4C, and 5C. b. Add buffer into the cuvettes Add de-ionized water. c. Add substrates just before start of the experiment. d. Spectrophotometric readings are taken. e. This protocol is basic and needs to be adjusted to study the effect of different factors on rate of reaction. To test dependency on temperature: a. 4 experimental cuvettes having the same experimental concentrations are taken.4 control cuvettes are also taken. They are prepared according to basic protocol. b. We shall test 4 different temperatures- 4 degrees (ice-bath), 15 degrees, 37 degrees and 50 degrees. c. After the cuvettes were prepared they are incubated at different temperatures. The control tubes acts as control for each set of experimental cuvettes where enzyme is added later after addition of buffer. d. The absorbance is measured at 440nm e. Graph is plotted taking specific activity along Y axis and temperature on X axis. Test dependency on pH: 1. All experimental cuvettes will have same concentration but the buffer pH in each need to be different. 2. After proper preparation of cuvettes with appropriate and varying pH the cuvettes are incubated in room temperature. 3. Absorbance at 440nm was measured. Test to see effect of Substrate concentration: 1. For this experiment we will be using varying concentrations of PNPP substares- 0.05 M, 0.01 M, 0.005 M and 0.1 M of PNPP. 2. Cuvettes are prepared by addition of buffer, enzyme and substrate. 3. Absorbance is measured at 440nm. Conclusion: The main of the experiment was to understand what effect different temperatures, pH and concentration have on the enzyme activity of the enzyme alkaline phosphatase. The results reveal that like every enzyme even for alkaline phosphatase there exists optimum conditions in which the specific activity of the enzyme is highest while the activity decreases in other conditions. 2.Biomolecules Types, Structure and Function of Carbohydrates, Lipids and Protein Carbohydrates, Lipids and Proteins are the key molecules of life. Let us look at their basic classifications, structures and functions. Carbohydrates: Carbohydrates are one of the major classes of biomolecules. Most of the earth’s organic matter is comprised of carbohydrates since they play an extensive role in the life of all organisms. They are compounds of carbon, hydrogen and oxygen. The carbohydrates are also known as saccharides because their basic component is sugar. Types and Structure and Function Chemically, carbohydrates are either aldehydes or ketone derivatives of alcohols with more than one –OH group.Carbohydrates are particularly of three types- Monosaccharide, disaccharides and polysaccharides. Monosaccharide- They are the simplest form of carbohydrates. They are basically aldehydes or ketones with one or more than hydroxyl groups attached. The empirical formula for monosaccharides is (C-H2O)n. Di-hydroxyacetone and d- and l- glyceraldehydes are the simplest form of monosaccharide for which the carbon number is three. They are known as triose. Monosaccharides with carbon numbers four, five six and seven are known as tetrose, pentose, hexose and heptose respectively. Of all monosaccharides trioses, pentoses and hexoses are of immense importance owing to their role in metabolism of cells. Disaccharides- Disaccharides are made up of 2 molecules of monosaccharides and are the simplest forms of polysaccharides. The two molecules are joined by an O-glycosidic bond. For examples, lactose, a disaccharide is constituted of galactose and glucose and is a major sugar found in milk. The three most abundant disaccharides is lactose, sucrose and maltose. Polysaccharides- More than 2 monosaccharides forms polysaccharides. Large polysaccharides may contain hundreds of monosaccharide units. If all the monosaccharides are same they are known as homopolymers. These polysaccharides can function as reservoir of glucose and form the structural unit for tissues. Glycogen, a homopolymer, is the most common storage molecule in the animal body is a long and branched polymer of glucose. Starch, is the nutritional reservoir in plants. Starch has two different forms- unbranched amylase and branched amylopectin. Medical disorder involving glucose High level of glucose in the blood stream gives rise to Type I diabetes. This is a chronic disease which can occur at any age. Beta cells of the pancreas produce insulin which controls the level of glucose in the body (Medline Plus, 2014). In people with this disease little or no insulin is produced thereby leading to high levels of glucose in the body. Normal treatment includes usage of insulin injections, monitoring of blood glucose, controlled diet and exercise (Daneman, 2006). Current disease management focus is upon usage of non-insulin adjunct therapy directed against the disease (George & McCrimmon, 2013). Lipids (triglycerides and phospholipids) These are a heterogenous group comprising of carbon, hydrogen and oxygen. Lipids are water insoluble and contain less proportion of oxygen.Cellular membranes are structurally made up of proteins floating in a sea of lipids. The lipid component of membrane is responsible for permeability of the membranes. Phospholipids- These are the basic building blocks of biomembranes. The physical properties of phospholipids enable it to form sheet-like structures. Phospholipids are basically made up of two long chains of non-polar fatty acyl groups linked to smaller highly polar groups such as phosphate usually by ester bonding. Phosphoglycerides are the major classes of phospholipids. In phosphoglycerides, the two fatty acyl chains is esterified to 2 of the 3 hydroxyl groups of glycerol while a phosphate is esterified to the third hydroxyl group. The simplest phospholipid is phosphatidic acid. In most phospholipids found in the membranes, the phosphate group is esterified to the hydroxyl group of a highly hydrophilic compound. For example in phosphatidylcholine, the choline group is bonded to a phosphate group. Figure1: phosphatidyl choline Triglycerides Triglycerides are a type of simple or storage lipids. Triglycerides are named so because of three fatty acids are attached to the glycerol molecule. Medical problem associated with lipids Dyslipidemia is the name given to abnormal level of lipids in the blood stream. The medical condition of importance since it is associated with several heart problems as well.Treatment with statins had proved futile and therefore researchers have focussed upon looking for better treatment options for patients suffering from this problem. Some of the alternatives that are currently being explored for lowering lipid level in the body include- development of antisense oligonucleotides targeting the production of apolipoprotein(apo)B-100, microsomal triglyceride transfer protein inhibitors, peroxisome proliferator-activated receptor agonists etc (Tavridou et al, 2011). Proteins Proteins are the most important working molecules of the cell. Broadly proteins are classified as structural and functional proteins. Structural proteins are responsible for providing structural rigidity to the cell while functional proteins help in carrying out various functions. Functional proteins are gain of various kinds – transport proteins, regulatory proteins, signalling and motor proteins. Structure of proteins Proteins are formed from polymerization of 20 different kinds of amino acids. Repeated amide, alpha carbon and the carbonyl group of each amino acid residue forms the backbone of the protein structure. This backbone shows directionality owing to the peptide linkages as all amino acids are located on the same side of the alpha carbon leaving an amino group free on one side of the protein chain and a free carboxyl group at the other end of the chain. Protein structure is of 4 categories- Primary, secondary, tertiary and quaternary. The primary structure of the protein is a simple linear arrangement of the amino acid residues where short chains are known as peptides (contains about 25-30 amino acid residues) while longer chains are known as polypeptides (may contain as many as 4000 residues). The secondary structure is a result of spatial arrangement of primary protein chains which have the ability to fold in some parts. A single polypeptide chain is capable of exhibiting more than just one secondary configuration depending on the sequence. Two well-defines secondary structures are – alpha helix and beta sheet structure of proteins. The alpha helix structure comprises of a segment of the polypeptide chain where the oxygen atom of carbonyl group is hydrogen bonded to hydrogen atom of the amide group located four residues away. Alpha helix holds a rod like structure where the amino acids form the backbone and the side chains point outwards. A alpha helix exhibits its hydrophobic or hydrophilic quality depending on the side chains since the backbone’s own polar groups are already bonded. Figure 2 :a. Ribbon depiction of alpha helix b.ball and stick model of alpha helix c.End view showing the coiled backbone d. Space filled showing tightly packed interior (Stryer et al, 2002, p 104) The Beta sheet is made up of beta strands that are packed laterally. Each strand is shorts and has about 5-8 residues. The sheet is formed by hydrogen bond formation between the backbone atoms of either the adjacent beta strands of the same polypeptide or between different polypeptides. Figure 3: A parallel beta sheet (Stryer et al, 2002, p 108). The Tertiary structure of proteins refers to the three dimensional arrangement of amino acid residues. As compared to the secondary structure which is stabilized by hydrogen bonding, tertiary protein structure is stabilized by hydrophobic interactions that occur between non-polar side chains, peptide bonds and hydrogen bonds. These hold the primary and secondary structures together. However owing to the weak stabilizing interactions, tertiary structures are prone to continual fluctuations. The quaternary structure is the most complex protein structure. It is 3 dimensional and made up of several peptides and polypeptides. Haemoglobin exhibits all four different structures. Basically it is composed of 2 alpha-globin and 2 beta-globin polypeptide chains. In primary structure the strands are joined together by peptide bonding. In secondary structure, alpha helixes of the primary are held together by hydrogen bonds. In tertiary, haemoglobin’s globular structure is 3 dimensional and held together by hydrogen and ionic bonds. In the quaternary configuration the four globular haemoglobin are held in a pyramidal shape around a central iron atom. Figure 4: The α2β2 Tetramer of Human Hemoglobin References Daneman, D. (2006).”Type 1 Diabetes.Lancet, 367 (9513), p.847-858. George,P. And McCrimmon,R.J.(2013).” Potential role of non-insulin adjunct therapy in Type 1 diabetes”.Diabetic medicine: a journal of British diabetic association, 30 (2), p.179-188. Medline Plus. (2014).Type 1 Diabetes.U.S. natonal library of medicine. Retrieved from http://www.nlm.nih.gov/medlineplus/ency/article/000305.htm on 6 October, 2014. Tavridou,A. et al.(2011).”Emerging targets for treatment of Dyslipidemia”. Current Medical Chemistry, 18 (6), p.909-922. Pictures: Stryer et al.(2002).Biochemistry(5th edition). Retrieved from http://www.ncbi.nlm.nih.gov/books/NBK22550/ Read More