BIOL 1450 Lab 1 - 6.2 Practical Questions

(LAB 5) Conversely, there may be an upper limit to cell size. The cell membrane is the interface through which the living interior volume of the cell interacts with its surroundings. Describe the relation between cell size, cell volume, and cell membrane area, and suggest how this relation might limit the size of a cell. (See your textbook for a detailed discussion.)

As the volume of the cell increases, the surface area of the cell decreases. The surface area is needed to help with transportation of substances in and out of the cell. If the cell becomes too big, the substances will not be distributed to the areas of the cell that need them.

(LAB 3) Describe how hydrophilic molecules behave differently than hydrophobic molecules in aqueous (water) solution.

Hydrophobic molecules are "water repelling" molecules which makes them nonpolar and less soluble in water. This means that the atoms of water and the hydrophobic molecules are equally charged, so they are not attracted to each other. Hydrophilic molecules are "water loving" molecules which make them polar and more soluble in water. This means that atoms of water and hydrophilic molecules have partial charges which are attracted to each other.

(LAB 6-1) What did we predict will happen if our model of the reaction is correct?

If the model of the reaction is correct, then without the addition of OAA, there should not be any changes in absorbance.

(LAB 3) Not all biological molecules are water-soluble. Many molecules have properties that cause them to restrict their surface exposure to water. Consider the structures of oil (fat) and wax; what can you predict about their solubility in water from their structures?

Oil and wax will not be able to dissolve in water, because they are considered to be hydrophobic substances. The carboxyl group is heavily barricaded by the triglyceride chains.

(LAB 2) Using your experience in lab as a guide, explain the steps you would use to determine whether the spectrophotometer could be used to quantify a different organic molecule (other than the dye) in solution.

Water alone gives readings, so a different organic molecule may absorb light at a given wavelength. The key is to identify the wavelength your organic molecule absorbs light at. A different organic molecule, dissolved in water, may be quantified by first blanking with water at each wavelength followed by taking a spectrophotometer reading of your organic molecule

(LAB 6-1) What did we do to confirm that the consumption of the measured substrate is through concomitant consumption of the second substrate, as in our model?

The consumption of the measured substrate was confirmed through concomitant consumption of the second substrate by not including OAA, and then combining NADH with the enzyme and recording any changes resulting from the combination.

(LAB 3) What explains the resistance of the MSG molecule to pH change, relative to the water? What feature of the MSG molecule is responsible for this?

The resistance of the MSG molecule to pH change occurs because MSG accepts H+ ions when the H+ conc. is high and donates H+ when H+ conc. is low, so the ions don't get a chance to change the pH of the solution. MSG's behavior is that of a buffer; it is the feature of the MSG molecule that is responsible for keeping the concentration fluctuations of the H+ and OH- ions from changing rapidly in the solution.

(LAB 5) The endomembrane (secretory membrane) system of eukaryotic cells is distinct from other internal membrane systems. Identify the major components of the endomembrane system and identify the organelles of the endomembrane system that you might have been able to see during this exercise and the organism in which they occurred. Identify a non-endomembrane system organelle that you saw, and the organisms in which it occurred.

The nuclear envelope, rough and smooth endoplasmic reticulum, Golgi apparatus, vesicles and the plasma membrane are the major components of the endomembrane system. The human cheek, onion, and elodea plant cells showed very distinct plasma membranes and the nuclei.

(LAB 6-1) How does our model of the proposed reaction account for this change in absorbance?

Absorbance decreases when the enzyme is added. NADH is losing electrons.

(LAB 3) When dissolved material is caused to come out of solution, we say that it has "precipitated," or that it has "formed a precipitate." We observed that continued addition of acid (HCl) to the MSG solution eventually caused a precipitate to form. Given what you know about how molecules are dissolved in water, suggest an explanation for how it was that addition of acid (free, positively charged protons) caused the MSG to precipitate.

Addition of HCl to the solution will increase the concentration of H+ and cause the carboxyl to associate a proton. When the carboxyl is no longer charged, it becomes less hydrophilic and is less able to interact with water. This causes a decrease in the amount of MSG that will dissolve. The undissolved MSG aggregates and forms the precipitate

(LAB 3) Summarize the effect that addition of monosodium glutamate had on the solution in which it was dissolved. Specifically, how did MSG modify the solution's capacity for pH change, or its resistance to pH change? What comparison did we use to detect this change in the solution's properties?

Addition of MSG increased the pH of the solution and also made the solution resistant to pH change. In other words, MSG behaved as a buffer. We tested this property by adding acid to the MSG solution and monitoring the pH. The pH change in the MSG solution upon acid addition was compared to the pH change in water upon acid addition to detect the difference in the solution's properties

(LAB 6-1) Use the space here to recount the relation between the information content of the genes (in DNA) and the functional protein that may be an enzyme (the central dogma) and To the far right side of the diagram (the central dogma), add a final feature—the "activity" of the functional enzyme protein.

DNA is transcribed into mRNA, which is taken out of the nucleus. The ribosomes in the cytosol translate mRNA to make proteins. These proteins could be enzymes that regulate metabolic activity. DNA transcribed to mRNA translated to Protein Regulation by Protein Enzyme

(LAB 3)Though the MSG—saturated solution had already reached its limit in dissolving MSG, did this prevent it from also dissolving sucrose?

Even though the MSG-Saturated solution reached its limit in dissolving MSG, it was not prevented from dissolving sucrose.

(LAB 2) Given the absorption spectrum of the cuvette containing water, explain why it is necessary to "blank" the spectrophotometer at each wavelength prior to taking an absorption measurement of a sample. What could happen if you did not blank the spectrophotometer after changing the wavelength?

It is important to "blank" the spectrophotometer before taking an absorption measurement of a sample at each new wavelength, because the water and cuvette also absorb light, so the spectrophotometer won't measure the absorption of water and cuvette. If the spectrophotometer is not "blanked", then it will read and add the absorption measurement of water and cuvette to the measurement of the dye. The desired result is to find out the absorbance of the dye and not water and cuvette.

(LAB 3) How did we determine that the MSG solution was saturated? Did the fact that the solution was saturated with MSG mean that it was incapable of dissolving other materials? How did we test this?

The MSG solution was saturated by observing the indissoluble MSG particles concentrated in the center of the bottom of the beaker. The solution proved that even though it was saturated with MSG, the solution was able to dissolve other materials. This was tested by adding 0.5g of sucrose to the solution.

(LAB 6-1) Had the absorbance changed following the addition of enzyme in the presence of all substrates?

The absorbance changed and decreased with the addition of an enzyme.

(LAB 6-1) IF we can monitor changes in substrate concentration spectrophotometrically, and IF we put all of the substrates (NADH and OAA) together in a conducive environment with the active enzyme, THEN, what should we observe?

The absorbance levels should go down after NADH concentrations lowers once the enzyme is added.

(LAB 6-1) If our model for the enzyme reaction was wrong, what would be an alternative explanation for this change in absorbance?

The concentration of OAA and NADH decreased, and thus the change in absorbance also decreased.

(LAB 3) When looking at the chemical structure of a molecule, what things help you to predict whether the molecule will behave like a hydrophilic molecule or a hydrophobic molecule?

The chemical structure of a molecule contains information on the types of charges and ions present in the structure. Hydrophilic molecules usually have partial charges indicated, while hydrophobic molecules have no charges indicated in the chemical structure.

(LAB 5) Comparing the change in mass of potato disks incubated in water with that observed when the disks were incubated in saturated NaCl, explain the basis for the difference between the treatments in terms of water movement as dictated by solute concentration.

The difference between the water movement in the water solution and salt solution is that the salt solution had a highly concentrated solute in the solution. Water moves from an area of low concentration of solute to an area with high solute concentration, so the mass of the potato disks in the salt solution decreased from 0.87g to 0.70g, because the water in the potato disks moved to where salt was concentrated the most, which is outside of the cell. The water solution did not have any solutes to which the water will be attracted to, so the mass of the potato disks in the water solution stayed at 0.80g and did not change.

(LAB 4) Write the hypotheses you developed in the laboratory regarding the amount of protein in membrane and soluble fractions in leaf and root. Write an analysis of your data to report whether your results support your hypotheses.

The first hypothesis developed in the laboratory regarding the amount of protein in membrane of the leaf and root is that the leaf will have more membrane-bound cell proteins than the root. The second hypothesis is that the leaf soluble proteins will have a small concentration compared to the root soluble proteins. The first hypothesis is supported by the leaf membrane protein concentration measure of 0.5128u/ug which is greater than that of the root membrane protein concentration of 0u/ug. The second hypothesis is not supported due to the leaf soluble proteins having a concentration of 0.2613u/ug, which is greater than the root soluble's concentration of 0u/ug. The reason for root soluble and root membrane proteins having a concentration measure of 0u/ug is for human errors possibly completed in the lab. The errors may have consisted of not pipetting the right amount of substances, forgetting to stir the solutions, not waiting for the dye to interact with the substances. Additional errors also resulted from not being able to grind the root completely or accidentally delivering extra material into the centrifugal test tubes. Data obtained from the experiment concerning the root soluble and root membrane may not be accurate, but the root also may not have had enough proteins present for the experiment. According to this experiment, however, the leaf membrane concentration is the highest of the four samples.

(LAB 3) Given your understanding of the role of molecular surface charge, explain how the addition of protons (in the form of hydrochloric acid) brought about this change: what has the presence of large quantities of protons in the solution done to the carboxyl groups of the molecule, and what effect has this had on its solubility?

The presence of large amount of proteins inhibits the solubility as an effect, because the vast amount of proteins are attracted to/form bonds with the carboxyl groups of the molecule, so the water molecules are unable to form bonds with the carboxyl groups.

(LAB 6-1) During this lab we tried to measure the light absorbance of solutions containing different concentrations of oxaloacetate, malate, and NADH. Explain the purpose behind these measurements. Specifically, what they were meant to discover that would apply to our development of an assay for measuring the activity of the enzyme? Explain why we would want to see a relation between the concentration of the substrate and its absorbance, as measured by the spectrophotometer.

The purpose of measuring the light absorbance of solutions containing various concentrations of oxaloacetate, malate, and NADH was to determine whether the spectrophotometer could be used to determine the differences between the differing concentrations of substrates or not. A visible relation between the high concentration of the substrate and its high absorbance is favorable, because the goal is to determine which substrate could be used to serve as a device to figure out if the consumption of the substrate could be detected in the reaction.

(LAB 6-1) At the end of the lab, you were asked to consider how the rate of NADH consumption would is related to the amount of enzyme added to the reaction assuming our model for the reaction is correct. Write your prediction by responding to the question posed: if the consumption of NADH is linked to the consumption of OAA, and if this consumption is catalyzed by an active enzyme, then what can we predict about the rate of NADH consumption relative to the amount of enzyme added to the reaction in the cuvette?

The rate of NADH consumption will be increased due to the greater presence of an enzyme which increases the rate of reactions.

(LAB 6-1) IF the consumption of NADH is linked to the consumption of OAA, and IF this consumption is catalyzed by an active enzyme THEN what can we predict about the rate of NADH consumption relative to the amount of enzyme added to the reaction in the cuvette?

The rate of NADH consumption will increase as the enzyme concentration will also increase.

(LAB 2) Describe the shape of the distribution of bean mass measurements around the calculated mean. Where do the majority of individual measurements occur, relative to the mean value? Note the overall jaggedness (with gaps and peaks) of the distribution when using a limited number of measurements. How would this distribution appear if you used fewer observations - would it be more jagged or less? Describe what you think would happen to the shape of the distribution if it were generated from one thousand, or one million observations.

The shape of the distribution of bean mass measurements around the calculated mean looks like a jagged bell curve. The majority of individual measurements occur at the 0.37g to the left or under the mean of .38g with the highest value of nine cranberry beans. The fewer number of observations used in the distribution the more the graph will be jagged, because there is not enough data to spread out evenly. If more observations were to be added the graph would show a more defined or smooth bell curve. It would also show more accurately or evenly distributed values.

(LAB 6-1) Did it appear that we could use the spectrophotometer to measure the concentration of any of these substrates or products? Which?

The spectrophotometer could be used to measure the concentration of NADH.

(LAB 2) You measured the absorbance of a dilute solution of indigotine at the same intervals across the spectrum and you blanked the spectrophotometer at each wavelength prior to measuring the absorbance of the dye sample. Why?

The spectrophotometer was "blanked" at each new wavelength, so the water and cuvette absorption measurements would not interfere with the dye absorption measurements. The absorption of indigotine solution was measured at the same intervals, because the interval chosen suited best for measurement of the solution due to having a high absorbance value.

(LAB 5) Given the nature of the molecules that make up cells, the structures built from these molecules, and the functions these molecules carry out, speculate whether there is a limit to how small a living cell could be. What factors would contribute to this limit?

There is a limit to how small a living cell can be. The living cell cannot be smaller than the organelles it contains due to organelles not being able to reside in the space and carry out their functions. Structure correlates with function, so the cell can't physically be smaller than the proteins it was built out of, because, if it is, then there must have been a problem in the mRNA message.

(LAB 6-1) Explain what was observed when the reaction mixture included active enzyme. Distinguish between what you saw, and what you understood the observation to mean was happening. According to our model of what occurs in the reaction, what other unseen events were also occurring at the same time? Explain the significance of attempting the reaction without the inclusion of OAA.

When the active enzyme was added to the reaction mixture, one observation made was that the absorbance levels were dropping rapidly until a constant rate of absorption was formed after the thirty second mark to about three units. The observation determined that the constant dropping of absorbance was boosted by the enzyme, so the enzyme increased the rate of the reaction as the substrates were being used up in the process. The unseen events occurred at the same time were electrons and the proton being taken away from NADH as OAA was being consumed in the process as NAD+ and malate were formed. Without OAA, malate would not be formed, and there would be nothing for the electrons to be transferred to for the change to malate to happen.