STUDY GUIDE TO LAB EXAM 2: USE THE STUDY GUIDE TO EXAM 1 TO REVIEW.

LABORATORY 5: MEASUREMENT OF ENZYME ACTIVITY

The study of enzyme activity plays a central role in our attempts to understand the molecular mechanisms of biological systems. Enzyme catalyzed reactions proceed through an intermediate that is expressed in the following equation. Equation 1.

E = the enzyme S = the substrate of the enzyme ES = the enzyme-substrate complex P = the product of the reaction kn = rate constants that describe the rate at which each of the reactions indicated by the arrows proceed. B. Measurement of enzyme kinetics The rate of enzyme reaction is referred to as the velocity of the reaction. It is most common to express the velocity in amount of product formed per minute. The initial reaction velocity, v0, of an enzymatic reaction varies with the substrate concentration, [S]. This can be illustrated by plotting v0 vs. [S] (figure 2 of lab manual). This plot is known as a Michaelis-Menten plot. It was named for two pioneers in the field of enzyme kinetic analysis. From this plot, an equation can be derived to describe the characteristics of an enzyme catalyzed reaction. The equation is called the Michaelis-Menten equation. The common form of the equation is v0= Vmax[S]/(Km + [S])

Equation 2. v0 = initial reaction velocity. The v0 is expressed as amount of product produced per unit time. The units of v0 are M/min or moles/min. Vmax = maximal reaction velocity; acheived when all the enzyme active sites are occupied with substrate molecules. This condition is called substrate saturation. [S] = substrate concentratation Km = Michaelis constant = (k2 + kp)/k1 Two important values, Vmax and Km can be determined from the graph shown in figure 2. The Vmax is the maximal rate at which an enzyme catalyzes a reaction. This value can be extrapolated from the plateau of the curve on the MichaelisMenten plot. The plateau region represents reaction conditions in which the concentration of substrate far exceeds the concentration of enzyme active sites. Therefore, all active sites will be occupied at any given time. The enzyme is said to be saturated with substrate under these conditions. The Vmax is not a constant because it depends on the amount of enzyme in the reaction. It is a very useful number because it can be used to calculate a constant called the turnover number, kp. The turnover number, kp, is the number of substrate molecules transformed to product by one enzyme molecule per unit time. It can be calculated from Vmax using the equation.

kp = Vmax/[Et] Equation 3
[Et] = the total concentration of enzyme in the reaction. The units are mg/ml or molarity (M). The turnover number is useful because it is a measure of the efficiency of the enzyme. The higher the kp, the "better" the enzyme. The Km or Michaelis constant is the other very useful term that is derived from the Michaelis-Menten equation. As is shown in the key to the Michaelis-Menten equation, the Km is related to the rate constants of the reaction. In simple terms, it is the ratio of the rate at which ES forms (k1) to the rate at which ES dissociates (k2+kp). The dissociation of ES is the sum of the rate that the substrate dissociates from the enzyme before it

undergoes the enzyme catalyzed reaction (k2) and the dissociation of product following the reaction (kp). There are three important points to remember about Km. 1) The Km is a measure of the apparent affinity of the substrate for the enzyme. The substrate with the lowest Km value has the highest apparent affinity for the enzyme. The "best" substrate is that which has the highest Vmax/Km. 2) Km is directly related to Vmax. It represents the substrate concentration at 1/2 the maximal velocity (1/2 Vmax) of the enzyme. 3) The Km of an enzyme is a constant for every individual enzyme with a particular substrate. Therefore, it serves as a fingerprint for the presence of a specific enzyme. This is very useful for investigators who are determining whether an enzyme is present or absent in a particular cell or tissue. The units of Km are molarity, M. The fact that the Michaelis-Menten plot gives a curve makes it difficult to accurately determine the Vmax and Km values from a graph. Therefore, the terms in equation 2 are often rearranged and used to generate a plot that gives a straight line. This equation is called the Lineweaver-Burk equation.

Equation 4 Equation 4 This equation is in the form, y = mx + b. By plotting 1/v0 versus 1/[S], this equation gives a straight line. This is illustrated in figure 2. The intercept on the 1/v0 axis is 1/Vmax and the intercept on the 1/[S] axis is -1/Km. The slope of the line on the plot is Km/Vmax.

Figure 2. Lineweaver-Burk plot for an enzyme-catalyzed reaction.

C. Experimental procedures Determining the activity and kinetic properties of an enzyme, wheat germ acid phosphatase. Acid phosphatase catalyzes the hydrolysis of phosphate groups from proteins, nucleic acids and lipids that are stored in the seed. To measure its activity, you used a substrate called p-nitrophenol phosphate. This substance is colorless. However, it is hydrolyzed by acid phosphatase to yield phosphate + nitrophenol. The nitrophenol is yellow. The amount of yellow

color generated by the catalysis is a direct measure of the amount of nitrophenol produced and therefore, is an indicator of the enzyme activity. In the first exercise, you measured the velocity of the reaction catalyzed by acid phosphatase that is extracted from wheat germ. In addition measured the velocity of purified acid phosphatase as a standard for the enzymes activity. By comparing the two values, you determined the amount of enzyme which is present in the wheat germ extract. FOR THE EXAM, BE ABLE TO USE SIMILAR DATA to determine acid phosphatase levels in wheat germ extract You added 5 ml of phosphatase substrate solution to tube A and 5 ml to tube B. Tube A contained the reaction catalyzed by purified acid phosphatase. Tube B contained the reaction catalyzed by the extracted acid phosphatase. At the times indicated, you removed 0.5 ml of the solutions in tube A and B and place them into the corresponding tubes containing KOH, and recorded the absorbance. The KOH in the numbered tubes stopped the reaction because the acid phosphatase is inactive at alkaline pH (hence the name acid phosphatase) and also turned the liberated nitrophenol to a more intense yellow. This made the assay more sensitive. Six standards containing known concentrations of nitrophenol were used to quantitate the concentrations of nitrophenol generated in your enzyme assays. Nitrophenol standard 1 2 3 4 5 6 Tube Nitrophenol (mmolar) Absorbance C1 C2 C3 C4 C5 C6 0 12.5 25 50 100 200

You read the absorbance of each of the standards at 410 nm with the spectrophotometer. Using this data you plotted your nitrophenol standard curve.

Using the nitrophenol standard curve, you determined the concentration of nitrophenol produced (mmolar) in each assay tube (A1-A6 and B1-B6). You then plotted the concentration of nitrophenol produced (Y axis) against time (X axis) for your samples in set A and B. From the graph, you calculated the initial velocities (v0) for the two reactions in mM/min. The v0 that you calculated actually represents the Vmax for the enzyme because the amount of p-nitrophenyl phosphate added to each reaction was at a concentration that saturates the enzyme. Given the fact that you added 2.5 mg of purified acid phosphatase to tube A, calculate the amount (mg) of acid phosphatase that was present in the 0.2 ml of wheat germ extract in tube B. HINT: To calculate the amount of enzyme in the wheat germ extract, you must first convert the Vmax from the purified enzyme to a constant that can be applied to the enzyme in the wheat germ extract. This constant is the turnover number. This constant can then be used to calculate the amount of enzyme in the wheat germ extract. 5. B. Determination of Vmax and Km for Acid Phosphatase You used the information on v0 that you obtained above to determine the Vmax and Km of purified acid phosphatase. This was a double-check of the Vmax that we assumed in the first part of the laboratory. First, you chose a time point in the enzyme assay that lies within the linear range of the initial velocity. Second, you set up a set of acid phosphatase assays containing varying amounts of substrate, but a constant amount of enzyme. After incubating the assays for the predetermined time, You quantitated the concentration of nitrophenol produced (mmolar) in each reaction using the standard curve from step 10 in the previous exercise. GIVEN SIMILAR DATA on the exam, you should be able to do the following: Convert these values to v0 values by dividing the concentration of nitrophenol produced by the reaction time. Prepare a Michaelis-Menten plot with the above data.

Estimate the Km and Vmax values from this plot. Express the values in the units of mM and mM/min, respectively. Prepare a Lineweaver-Burk plot of 1/v0 versus 1/[S]. Determine the Km and Vmax from the Lineweaver-Burk plot. Are the values different from those that you estimated from the Michaelis-Menten plot? LAB 6:GEL FILTRATION CHROMATOGRAPHY We will express the size of a protein as "molecular weight" (Mr, relative molecular mass), which is based on a ratio of the mass of a molecule to 1/12 of the mass of carbon 12. The value of Mr is unitless because it is the ratio of two masses.

The two most common methods for protein molecular weight determination are gel filtration chromatography and sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Both rely on the movement of proteins through a porous matrix to distinguish proteins of different sizes. Both methods will give an estimated or apparent molecular weight for a protein. The actual molecular weight of a protein can only be deduced from its primary sequence by adding up the masses of its component amino acids. The estimated and actual Mr for a protein can differ depending on the shape and physical properties of the molecule.

GEL FILTRATION CHROMATOGRAPHY Gel filtration chromatography is a form of column chromatography. It consists of a solid stationary phase and a liquid mobile phase. The stationary phase is confined to a column (glass tube). The substance that makes up the stationary phase is called the gel matrix. The gel matrix is composed of tiny beads made from highly cross-linked polymers, which swell in the presence of solvent to form microscopic porous sponges. In our case, the solvent will be a buffer. The porous nature of the gel matrix forms the basis for the separation method. The matrix that is packed into the column is often referred to as the column bed.

The liquid mobile phase (a buffer or solvent) flows through the solid stationary gel matrix phase. Protein in the mobile phase will interact with the matrix as it moves through the column. The degree of interaction will depend on the properties of the protein. Proteins interact with the matrix based on their molecular weight. A sample containing a mixture of proteins of different sizes flows into the matrix. As the mobile phase exits the bottom of the column, it is collected as aliquots of constant volume. The exiting mobile phase is called the column eluate, and each successive portion of the eluate is called a column fraction. The volume of the mobile phase that is required to elute a particular protein is called its elution volume (Ve).

. A linear relationship is obtained if the logarithms of the molecular weights of standard proteins are plotted against their respective elution volumes. The molecular weight of the unknown protein can be estimated by extrapolating from the standard graph.

Gel filtration chromatography will give you the apparent molecular weight of the native protein. The proteins do not undergo any harsh treatments prior to gel filtration chromatography. Therefore, they maintain their secondary, tertiary, and quaternary structure. This property of the method makes it very valuable for characterizing and purifying proteins whose functional properties (i.e. enzymatic activity) will be studied in subsequent experiments.

STANDARDS AND UNKNOWNS Myoglobin - Myoglobin is composed of a single polypeptide chain that is bound to a single prosthetic group called heme. The iron in the heme group functions in binding oxygen

Hemoglobin - Hemoglobin is a tetramer (i.e. it contains four polypeptide subunits) that associate by non-covalent interactions. The subunits are called alpha and beta and are of nearly identical molecular weight. The color of hemoglobin depends on whether it contains bound oxygen, but it usually is red to red/brown.

Given Ve and Mr of a few standards, you should be able to plot the Ve vs. log Mr using graph paper and produce a standard graph and use it to determine the molecular weight (Mr) of an unknown such as myoglobin or hemoglobin. LAB 7: SDS PAGE The method of electrophoresis is based on the fact that charged molecules move in an electric field. In the process of polyacrylamide gel electrophoresis (PAGE), proteins are placed in an electric field and are forced to move through a porous matrix or gel (polyacrylamide) by the current.

Unlike the beads used in gel filtration, the polyacrylamide is a continuous matrix that consists of only the polymer and pores. There is nothing equivalent to the void volume of a gel filtration column. To move through the gel, the proteins must move through the pores of the polyacrylamide.

PAGE also differs from the gel filtration column, in that the rate at which proteins move through is dependent on both their mass and their charge. Proteins that carry a high degree of charge will move at a faster rate than those of the same mass with a lower overall charge. Therefore, the mobility of the protein in PAGE will not be proportional to its size and cannot be used to estimate its molecular weight.

To eliminate this problem, proteins are treated with a detergent called sodium dodecyl sulfate (SDS). SDS is a detergent that binds to proteins and masks their native charged groups. Therefore, electrophoretic separation of SDStreated proteins sorts them according to size alone. This method is called sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins that are treated with SDS do not retain their native secondary, tertiary, or quaternary structure and are referred to as denatured proteins. Therefore, proteins that have quaternary structure will be dissociated into their constituent subunits and SDS-PAGE will reveal the Mr of the subunits, but not the Mr of the native protein. In addition, any prosthetic groups that are not covalently attached to the protein will be dissociated. Therefore, biochemists use a combination of gel filtration chromatography and SDS-PAGE to determine the Mr of a protein and its subunit composition.

The SDS-PAGE exercises Given a set of proteins of known molecular weight to use as standards, and their migration distances, you should be able to make a standard graph of distance vs log of molecular weights. You should also be able to use it to calculate the molecular weight of unknowns such as myoglobin and hemoglobin, if you are given their migration distances. Compare the molecular weights of myoglobin and hemoglobin as estimated by the two methods used in these exercises (Lab 4 and 5). Are they the same? If not give an explanation. Why do hemoglobin and myoglobin lose their reddish color during SDS-PAGE analysis? S-PAGE analysis?

LABORATORY 8: HUMAN DNA FINGERPRINTING

Regions of human chromosomes that exhibit a great deal of diversity are termed "polymorphic" (meaning many forms). These are the basis for genetic disease diagnosis, forensic identification, and paternity testing. For example, DNA from chromosome 8 contains a polymorphic sequence: an insertion of a short nucleotide sequence, called Alu, within the tissue plasminogen activator gene. A particular Alu insertion element, called TPA-25, is present in some individuals but not others, so it is useful in human DNA fingerprinting. Polymerase chain reaction (PCR) is used to screen individuals for the presence of the TPA-25 insertion. polymerase chain reaction (PCR) amplifies a DNA sequence by the repetition of 3 basic steps: a) the reaction mixture is heated to high temperature (94oC) to denature the double-stranded DNA template into two complementary strands b) the temperature is lowered (58oC) to allow the oligonucleotide primers to anneal with their corresponding sequences on the single-stranded DNA template c) the temperature is raised (72oC) to the optimal temperature for the polymerization of the complementary DNA strand by the DNA polymerase. In this

step, the polymerase will use the annealed oligonucleotide primers to synthesize a strand of DNA that is complementary to the template. -These cycles of denaturation, annealing, and DNA synthesis are repeated many times. Because the products of one round serve as templates for the next round, each successive cycle doubles the amount of the desired DNA product. PROCEDURE: The source of template DNA for amplification is a sample of several thousand cheek cells obtained by saline mouthwash The cells are collected by centrifugation They are resuspended in a solution containing the resin "Chelex," which binds metal ions that would otherwise interfere with the PCR reaction. The cells are lyzed by boiling The supernatant containing chromosomal DNA is mixed with Taq DNA polymerase, oligonucleotide primers, the four deoxyribonucleotides, and the cofactor magnesium chloride. PCR temperature cycling is used to denature the target DNA, anneal the primers, and extend a complementary DNA strand. The size of the amplification product(s) depends on the presence or absence of the Alu insertion at the TPA-25 locus on each copy of chromosome 8. Oligonucleotide primers amplify a 400-bp fragment when TPA-25 is present and a 100-bp fragment when it is absent. There are three possible genotypes 1) homozygotes for presence TPA-25 (400-bp fragment only), 2) homozygotes for absence of TPA-25 (100-bp fragment only), 3) heterozygotes (400-bp and 100-bp fragments) Each of these can be distinguished following electrophoresis in agarose gels. Aliquots of the amplified samples from students are loaded into wells of an agarose gel. Following electrophoresis and staining, amplification products appear as distinct bands in the gel - the distance moved from the well is inversely proportional to the presence or absence of the TPA-25 insertion.

Either one or two bands are visible in each lane, indicating that an individual is either homozygous or heterozygous for the Alu insertion. INTERPRETING RESULTS: a. No bands visible: an error during sample preparation, such as losing the cell pellet or using a too-acidic Chelex solution. b. One band visible. Compare its migration to those from other individuals on the photo to establish whether it corresponds to the 400 bp or 100 bp PCR product. Remember, the 400 bp band migrates slower than the 100 bp band and is therefore located closer to the sample wells. If only the 400 bp band is present, then that individual is homozygous for the TPA-25 Alu insertion (+/+). If only the 100 bp band is present, then that individual is homozygous for the absence of the TPA-25 Alu insertion (-/-). c. Two bands visible. This individual is heterozygous for the TPA-25 Alu insertion (+/-). d. Three or more bands visible. The one or two bright bands are likely the true alleles. Additional bands may occur when the primers bind nonspecifically to chromosomal loci other than TPA-25 and give rise to additional amplification products.significance of results: You would have been able to determine the genotype distribution for the class by counting how many students are (+/+), (+/-), and (-/-).