Quiz : Biochemistry Lab Questions And Answers

In the presence of a noncompetitive inhibitor, the Vmax (maximum velocity) of the enzyme-catalyzed reaction is affected. Noncompetitive inhibitors bind to the enzyme or the enzyme-substrate complex at a site other than the active site, causing a change in the enzyme's conformation and reducing its catalytic activity. This results in a decrease in the rate of the reaction, leading to a lower Vmax. The Km (Michaelis constant) is not affected by noncompetitive inhibitors as they do not compete with the substrate for binding to the active site. Therefore, the correct answer is Vmax.

The presence of glycerol in the sample buffer increases the density of the protein sample. This increased density helps prevent the proteins from diffusing out of the sample well during electrophoresis. Therefore, the statement is true.

An international unit of an enzyme is defined as the amount that catalyzes the formation of one micromol of product in one minute. This means that the enzyme is able to convert one micromol of substrate into product within a time frame of one minute. This unit is commonly used to measure the activity of enzymes and allows for standardized comparisons between different enzyme preparations.

The liver is responsible for producing most of the blood cholesterol in the body. It synthesizes cholesterol and releases it into the bloodstream to be transported to various tissues. The pancreas, heart, and kidney do not have a significant role in cholesterol production.

The term "blotting" refers to the transfer of biomolecules to an immobilizing membrane. This technique is commonly used in molecular biology to separate and identify specific proteins or nucleic acids from a mixture. The biomolecules are first separated by size using gel electrophoresis and then transferred onto a membrane, such as nitrocellulose or PVDF. The transferred biomolecules can then be detected using specific probes, such as antibodies or nucleic acid probes, allowing for further analysis and identification.

SDS-electrophoresis is a technique used to separate proteins based on their size. It involves denaturing the proteins with SDS (sodium dodecyl sulfate) which coats them with a negative charge, causing them to migrate towards the positive electrode. Since the migration rate depends on the size of the protein, smaller proteins move faster and travel further through the gel than larger proteins. Therefore, the separation of proteins by SDS-electrophoresis is based on the differences in protein size.

Most proteins have a net charge that is dependent on the pH of their environment. At a pH above 7.0, proteins tend to have a negative charge due to the deprotonation of their amino acid side chains. Therefore, the statement that most proteins have a positive charge above pH 7.0 is false.

A Lineweaver-Burk analysis is a graphical method used to determine the values of Km (Michaelis constant) and Vmax (maximum velocity) in enzyme kinetics. It involves plotting the reciprocal of the initial reaction rate (1/V0) against the reciprocal of the substrate concentration (1/[S]). The slope of the line represents Km/Vmax, while the y-intercept represents 1/Vmax. By analyzing the Lineweaver-Burk plot, both Km and Vmax can be determined accurately.

The proper component sequence in western blotting is as follows: fiber pad, filter paper, gel, nitrocellulose membrane, filter paper, fiber pad. This sequence allows for the transfer of proteins from the gel onto the nitrocellulose membrane, which is an essential step in western blotting. The fiber pads help to maintain even pressure during the transfer process, while the filter papers act as wicks to draw the transfer buffer through the gel and onto the membrane.

Enzymes increase the rate of reaction by decreasing the energy of activation. The energy of activation is the energy required for a chemical reaction to occur. By lowering this energy barrier, enzymes allow the reaction to proceed more easily and quickly. This is achieved by providing an alternative pathway with a lower activation energy, allowing more reactant molecules to reach the transition state and form products. Therefore, enzymes effectively decrease the energy of activation and enhance the rate of the reaction.

In Western blotting, the mobility of protein bands is actually from cathode (negative pole) to anode (positive pole). This is because proteins are negatively charged and are attracted towards the positive electrode. Therefore, the statement that the mobility is from anode to cathode is incorrect.

During gel electrophoresis, proteins are separated based on their size and charge. Smaller proteins can move more easily through the gel matrix and therefore have a larger Rf value, indicating that they have traveled a greater distance from the starting point. On the other hand, larger proteins experience more resistance and are not able to move as far, resulting in a smaller Rf value. Therefore, smaller proteins have a larger Rf value while larger proteins have a smaller Rf value during gel electrophoresis.

Lysozyme is an enzyme that hydrolyses glycosidic bonds. Glycosidic bonds are a type of bond that connects sugar molecules in carbohydrates. By hydrolyzing these bonds, lysozyme breaks down carbohydrates and helps in the defense against bacterial infections. Therefore, the correct answer is glycosidic bond.

The velocity of an enzyme-catalyzed reaction can be affected by various factors including enzyme concentration, substrate concentration, pH, and temperature. Enzyme concentration refers to the amount of enzyme present, which can influence the rate of reaction. Substrate concentration, on the other hand, refers to the amount of substrate available for the enzyme to bind to and catalyze. pH can affect the shape and activity of the enzyme, thus impacting the reaction rate. Temperature can also affect the rate of reaction as enzymes have an optimal temperature at which they work most efficiently. Therefore, all of these factors can affect the velocity of an enzyme-catalyzed reaction.

An RNA blot refers to a technique known as northern blotting. It is used to study gene expression by detecting and analyzing RNA molecules. Similar to a western blot that detects proteins, a northern blot specifically detects RNA molecules. This technique involves separating RNA molecules by size using gel electrophoresis, transferring them onto a membrane, and then hybridizing the membrane with a labeled RNA probe to visualize the target RNA of interest.

Cholesterol cannot be easily metabolized to CO2 and H2O in humans and excreted. While some cholesterol can be broken down and eliminated from the body, the majority of it is actually synthesized in the liver and used for various functions in the body. Excess cholesterol can build up in the arteries and lead to health problems such as heart disease. Therefore, it is important to manage cholesterol levels through a healthy diet and lifestyle.

The expression [S] at 1/2 Vmax is used for the determination of Km. Km is the Michaelis constant and it represents the substrate concentration at which the reaction velocity is half of the maximum velocity (Vmax). Therefore, measuring the substrate concentration ([S]) at 1/2 Vmax allows us to determine the Km value.

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a technique used to separate proteins based on their molecular weight. In this technique, proteins are denatured and coated with SDS, which imparts a negative charge to all the proteins. The proteins are then subjected to an electric field and migrate towards the positive electrode based on their size. Tris-buffer, b-Mercatoethanol, and heat are all necessary components for SDS-PAGE as they help in protein denaturation, maintaining pH, and breaking disulfide bonds. Ammonium persulfate (APS) is a reagent used to initiate the polymerization of acrylamide in the gel, and it is not directly involved in protein separation.

Acrylamide is the correct answer because it is a highly toxic chemical that is commonly used in gel electrophoresis. It can be absorbed through the skin and has the potential to cause central nervous system paralysis. Ammonium persulfate, TEMED, and SDS are not known to be extremely toxic in gel electrophoresis.

When [S] (substrate concentration) is much greater than Km (Michaelis constant), it indicates that the enzyme is saturated with substrate. This means that all the enzyme's active sites are occupied by substrate molecules, and any further increase in substrate concentration will not have a significant effect on the reaction rate. Thus, the enzyme activity remains constant at its maximum rate, and the reaction is said to be in the saturated state.

Enzymes decrease the energy of activation in enzyme-catalyzed reactions. Enzymes are biological catalysts that speed up chemical reactions by lowering the energy barrier, or activation energy, required for the reaction to occur. They achieve this by binding to the reactant molecules, bringing them closer together, and providing an alternative reaction pathway with a lower energy of activation. This allows the reaction to proceed more quickly and efficiently. Therefore, enzymes play a crucial role in facilitating biochemical reactions in living organisms.

pNPP (p-nitrophenyl phosphate) was used as a substrate during acid phosphatase kinetic analysis. This is because acid phosphatase is an enzyme that catalyzes the hydrolysis of phosphate esters under acidic conditions. pNPP is a commonly used substrate for acid phosphatase assays as it can be converted into p-nitrophenol by the enzyme, which can be measured spectrophotometrically. The rate of p-nitrophenol formation is directly proportional to the activity of acid phosphatase, allowing for the determination of enzyme kinetics.

The Michaelis-Menten assumptions state that the E, S, and ES (enzyme, substrate, and enzyme-substrate complex) are in rapid equilibrium, and the product formation is directly proportional to the ES complex concentration. However, the assumption that does not apply is that the product formation is inversely proportional to the ES complex concentration.

Methanol is used in the transfer buffer of western blotting to dissociate the SDS (sodium dodecyl sulfate) from proteins. SDS is commonly used in protein denaturation and electrophoresis, but it can interfere with the transfer of proteins from the gel to the nitrocellulose (NC) membrane. Methanol helps to remove the SDS, allowing the proteins to move more easily from the gel to the NC membrane during the transfer process.

The v/Vmax ratio represents the fraction of the maximum velocity (Vmax) that is achieved at a given substrate concentration ([S]). In this case, the substrate concentration is given as 3 times the Michaelis constant (Km). The Michaelis constant represents the substrate concentration at which the reaction rate is half of the maximum velocity. Therefore, if [S]=3Km, it means that the substrate concentration is three times higher than the concentration at which the reaction rate is half of Vmax. As a result, the v/Vmax ratio would be 0.75, indicating that the reaction is operating at 75% of its maximum velocity.

The electrophoretic pattern in SDS-PAGE under reducing conditions is primarily determined by the molecular weight of subunits. SDS-PAGE separates proteins based on their molecular weight, and reducing conditions break disulfide bonds, resulting in proteins being denatured and separated into subunits. The smaller subunits will migrate faster through the gel, while larger subunits will migrate slower, resulting in distinct bands on the gel. The other properties mentioned, such as the shape of the native protein, specific binding sites, and net charge, may also contribute to the electrophoretic pattern to some extent, but the molecular weight of subunits is the main determining factor.

Blocking step in western blot detection is used to decrease nonspecific binding. During the blocking step, a solution containing proteins such as bovine serum albumin (BSA) or nonfat dry milk is applied to the membrane. This solution fills up any unbound sites on the membrane, preventing nonspecific binding of antibodies or other detection reagents. By blocking nonspecific binding, the blocking step allows for more specific and accurate detection of the target protein of interest. Therefore, the correct answer is False.

An international unit of an enzyme is a standardized measure of its activity. In this case, it is defined as the amount of enzyme that catalyzes the formation of one micromol of product in one minute. This means that if the enzyme is able to produce one micromol of product in one minute, it is considered to have one international unit of activity. This definition allows for a standardized way of comparing the activity of different enzymes.

The statement is false because a polymerized gel is not toxic after polymerization. Polymerization is the process of converting a liquid monomer into a solid polymer network. Once the gel is polymerized, it becomes a stable and non-toxic material. The polymerized gel can be used as an ideal gel matrix for various applications due to its desired porosity and stability.

Lysozyme is an enzyme that works on the cell wall of Micrococcus lysodeikticus, hydrolyzing the glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid. This enzymatic activity is responsible for its ability to provide a defense mechanism against bacterial infection. Lysozyme is also found to be rich in tears. However, it does not hydrolyze phosphodiester bonds.

Km is a measure of the affinity between an enzyme and its substrate. It represents the substrate concentration at which the enzyme achieves half of its maximum velocity (Vmax). Therefore, Km gives an approximate indication of the substrate concentration within the cell. A lower Km value indicates a higher affinity between the enzyme and substrate, meaning that the enzyme can achieve half of its maximum velocity at lower substrate concentrations. Conversely, a higher Km value indicates a lower affinity between the enzyme and substrate, requiring higher substrate concentrations to achieve half of the maximum velocity.

The correct answer is Km. Km is a measure of the affinity between an enzyme and its substrate. In the presence of a competitive inhibitor, the inhibitor competes with the substrate for binding to the active site of the enzyme. This increases the apparent Km value, as more substrate is required to achieve the same reaction rate. The Vmax value, on the other hand, remains unchanged in the presence of a competitive inhibitor. Ki refers to the inhibition constant, which is not directly related to changes in Km. Therefore, the correct answer is Km.

Bromophenol blue is commonly used as a tracking dye in SDS-PAGE. It is added to the sample before loading onto the gel to monitor the progress of the electrophoresis. The dye migrates at a predictable rate, allowing the researcher to track the movement of the proteins and determine when to stop the electrophoresis. It is easily visible due to its blue color and does not interfere with the separation of the proteins.

Polyacrylamide gels are prepared by polymerization of acrylamide monomer and N-N'-methylene-bis-acrylamide cross-linker in the presence of APS (Ammonium persulfate) and TEMED (Tetramethylethylenediamine). APS acts as a initiator for the polymerization reaction by generating free radicals, while TEMED acts as a catalyst to enhance the reaction rate. Both APS and TEMED are commonly used in gel electrophoresis to initiate the formation of the polyacrylamide gel matrix.

Acetyl CoA is the precursor for cholesterol synthesis. It is formed from the breakdown of glucose or fatty acids in the mitochondria. Acetyl CoA is then converted into mevalonate, which is the first step in the synthesis of cholesterol. Choline, lipoic acid, and malic acid are not directly involved in the synthesis of cholesterol.

pNPP (p-nitrophenyl phosphate) serves as a substrate for Acid phosphatase. Acid phosphatase is an enzyme that catalyzes the hydrolysis of phosphate esters, and pNPP is commonly used as a substrate to measure its activity. When pNPP is hydrolyzed by Acid phosphatase, it produces pNP (p-nitrophenol) as a product. The amount of pNP produced can be measured spectrophotometrically, allowing for the quantification of Acid phosphatase activity. Therefore, pNPP is the correct answer as it specifically serves as a substrate for Acid phosphatase.

The specific activity of an enzyme is a measure of its catalytic efficiency and is typically expressed as the amount of substrate converted per unit time per unit of enzyme concentration. In this question, the enzyme Muramidase hydrolyzed 0.2 mmol of cell wall in 5 minutes, and the concentration of the enzyme is 2.5 mg/ml. To calculate the specific activity, we divide the amount of substrate converted (0.2 mmol) by the time (5 minutes) and the enzyme concentration (2.5 mg/ml). This gives us a specific activity of 80 micromol/min/mg.

In competitive inhibition, the inhibitor binds to the free enzyme. This means that the inhibitor competes with the substrate for binding to the active site of the enzyme. When the inhibitor is bound to the enzyme, it prevents the substrate from binding and thus inhibits the enzyme's activity. This type of inhibition can be overcome by increasing the concentration of the substrate, as it increases the chances of the substrate outcompeting the inhibitor for binding to the enzyme.

The protein with a molecular weight (MW) of 2,000 daltons will be at the bottom of the gel in the SDS-PAGE system. In this system, smaller proteins migrate faster and move further down the gel compared to larger proteins. Since the protein with a MW of 2,000 daltons is the smallest among the given options, it will migrate the furthest and be at the bottom of the gel.

In enzyme kinetic analysis, the pH curve is bell-shaped. This means that as the pH of the environment changes, the activity of the enzyme also changes. At a certain optimal pH, the enzyme activity is at its maximum. As the pH deviates from this optimal value, the enzyme activity decreases. This bell-shaped curve is a characteristic feature of pH-dependent enzyme kinetics and is often used to study the effect of pH on enzyme activity.

The proper component sequence in western blotting is as follows: fiber pad, filter paper, gel, nitrocellulose membrane, filter paper, fiber pad. This sequence ensures that the gel is sandwiched between the filter papers, which helps in the transfer of proteins from the gel to the nitrocellulose membrane. The fiber pads act as wicks to facilitate the transfer of buffer and prevent drying out of the gel.

A prosthetic group is a tightly bound nonprotein part of an enzyme. Prosthetic groups are small molecules that are permanently attached to enzymes and are necessary for their proper functioning. They can be organic molecules such as vitamins or inorganic molecules such as metal ions. Prosthetic groups play a crucial role in enzyme catalysis and can participate in the enzyme's chemical reactions. They are specific to certain enzymes and are not present in all enzymes.

All of the statements are true. Lysozyme is an enzyme that provides a defense mechanism against bacterial infection by breaking down the glycosidic bond in bacterial cell walls. It is found in various sources, including egg white.

When the cofactor is removed from an active enzyme, the resulting protein is called an apoenzyme. The cofactor is a non-protein molecule that is necessary for the enzyme to function properly. Without the cofactor, the enzyme loses its catalytic activity and becomes inactive. The apoenzyme can regain its activity when the cofactor is reattached, forming a holoenzyme. Therefore, the correct answer is apoenzyme.

The clear assay solution observed at the end of the reaction suggests that the enzyme, lysozyme, cleaves the glycosidic bond and disrupts the cell wall. This is because lysozyme is known to have the ability to break down the glycosidic bond found in the peptidoglycan layer of bacterial cell walls. When this bond is cleaved, it weakens the cell wall structure, causing it to become clear. The other options, such as cleaving hydrogen or disulfide bonds, do not have the same effect on the cell wall and would not result in a clear assay solution.

The Dixon plot is a graphical method used to determine the inhibition constant (Ki) in enzyme inhibition experiments. It involves plotting the reciprocal of the reaction rate against the inhibitor concentration, and the slope of the resulting line can be used to calculate the Ki value. This method is particularly useful for determining the type of inhibition (competitive, non-competitive, or uncompetitive) and provides a visual representation of the data, making it easier to interpret and analyze.

The initial velocity of a reaction can be calculated using the Michaelis-Menten equation, which is V0 = (Vmax * [S]) / (Km + [S]). In this case, Vmax is given as 150 micromol/sec and Km is given as 2mM. The concentration of substrate, [S], is given as 10 mM. Plugging these values into the equation, we get V0 = (150 * 10) / (2 + 10) = 1500 / 12 = 125 micromoles/sec. Therefore, the initial velocity at [S] = 10 mM is 125 micromoles/sec.

HDL, or high-density lipoprotein, binds plasma cholesterol and transports it to the liver. HDL is often referred to as "good cholesterol" because it helps remove excess cholesterol from the bloodstream and carries it to the liver for processing and excretion. This process helps to prevent the buildup of cholesterol in the arteries and reduces the risk of heart disease.

The colloidal gold reagent was used for total protein detection in the Western blot lab.