Why does high pH result in the denaturation of DNA?

Why does high pH result in the denaturation of DNA?

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In the Southern blot method, for example, a solution of NaOH is used to denature the DNA in the sample. I find this counterintuitive since I expected that $ ext{Na}^+$ cations would neutralize the negative charges of phosphate groups, hence stabilizing the double helix.

The pKas of (neutral) guanine and thymine are 9-10 (ref). At high pH (>~10), those bases will be deprotonated and exist as negatively charged conjugate bases. As the deprotonated species, part of the G/C and A/T hydrogen bonding networks are eliminated. In the figure below, green dotted lines represent the hydrogen bonds that explain the observed base pairing. Red dotted lines represent disrupted interactions.


Hyperchromicity is the increase of absorbance (optical density) of a material. The most famous example is the hyperchromicity of DNA that occurs when the DNA duplex is denatured. [1] The UV absorption is increased when the two single DNA strands are being separated, either by heat or by addition of denaturant or by increasing the pH level. The opposite, a decrease of absorbance is called hypochromicity.

Heat denaturation of DNA, also called melting, causes the double helix structure to unwind to form single stranded DNA. When DNA in solution is heated above its melting temperature (usually more than 80 °C), the double-stranded DNA unwinds to form single-stranded DNA. The bases become unstacked and can thus absorb more light. In their native state, the bases of DNA absorb light in the 260-nm wavelength region. When the bases become unstacked, the wavelength of maximum absorbance does not change, but the amount absorbed increases by 37%. A double stranded DNA strand dissociating to two single strands produces a sharp cooperative transition.

Hyperchromicity can be used to track the condition of DNA as temperature changes. The transition/melting temperature (Tm) is the temperature where the absorbance of UV light is 50% between the maximum and minimum, i.e. where 50% of the DNA is denatured. A ten fold increase of monovalent cation concentration increases the temperature by 16.6 °C.

The hyperchromic effect is the striking increase in absorbance of DNA upon denaturation. The two strands of DNA are bound together mainly by the stacking interactions, hydrogen bonds and hydrophobic effect between the complementary bases. The hydrogen bond limits the resonance of the aromatic ring so the absorbance of the sample is limited as well. When the DNA double helix is treated with denatured agents, the interaction force holding the double helical structure is disrupted. The double helix then separates into two single strands which are in the random coiled conformation. At this time, the base-base interaction will be reduced, increasing the UV absorbance of DNA solution because many bases are in free form and do not form hydrogen bonds with complementary bases. As a result, the absorbance for single-stranded DNA will be 37% higher than that for double stranded DNA at the same concentration.

Alkaline Lysis

Molecular biologists often make use of alkaline denaturation to isolate plasmid DNA from bacteria. Plasmids are little loops of DNA separate from the bacterial chromosome. In an alkaline lysis miniprep, biologists add detergent and sodium hydroxide to bacteria suspended in solution. The detergent dissolves the bacterial cell membrane while the sodium hydroxide boosts the pH and makes the solution highly alkaline. As the broken cells release their contents, the DNA inside separates into its component strands, or denatures.

2 Answers 2

The main component of egg white, ovalbumin, contains 93 negatively charged residues (Asp + Glu) and 68 positively charged residues (Arg + Lys), as well as 14 His. If you make the solvent less polar by adding ethanol to water, they will tend to form the neutral species (Arg+ and Lys+ will lose a proton, and Glu- and Asp- will pick up a proton).

If you had the protein unfolded already, you would expect that the solution gets more basic if the effect on all charged amino acids is the same because there are more negatively charged ones.

To test whether the observed effect has to do with denaturation of the proteins or with changes in protonation state of side chains already exposed to water, you could measure the pH of amino acids (preferably with a modification of the main chain carboxylic acid and amino functions so only the side chains show acid/base reactions) at different ethanol concentrations.

Most proteins are polyelectrolytes because the constituent aminoacids may have ionizable groups, typically carboxylic and basic (imino, guanidino) groups on the sidechains, in addition to the terminating amino and carboxylic groups (when not modified). The degree of ionization depends on the fold of the protein and the pH of the solution. Denaturation of a protein can expose to the solvent buried sidechains with ionizable groups, resulting in acid/base reactions with water.

If the solution is buffered and the protein is not present at high concentrations, then the degree of ionization of the protein will depend on the solution pH, but the solution pH will not be particularly sensitive to the presence of the protein. On the other hand, if there is a lot of protein in the solution and/or the solution is not buffered so as to resist changes in the degree of ionization of the protein, then such changes will translate in changes in pH. Concretely, changes in secondary or higher structure of the protein will translate into changes in pH, as it appears you observe.

Unfortunately (as noted in the comments), the measured pH of the sample may vary strongly with the concentration of ethanol even in the absence of protein and in the presence of buffer. This is in addition to the effect which the organic solvent will have on the dissociation constant of ionizable groups on the protein noted by Karsten Theis. It is quite likely that those effects will exceed in importance that of denaturation, since charged groups are most often exposed on the protein surface. The extent of exposure and the dissociation constant may increase with denaturation, of course, and disentangling the different effects is not simple, requiring accurate measurements.

To start with you might check the extent to which the addition of alcohol may be responsible for the increase in pH. A lot of work has already been done in measuring such effects, and the following table compiles some results reported in the IUPAC Compendium on Analytical Chemistry (Ref. 1) for the 0.05 m potassium hydrogen phthalate (KHPh) buffer.

Other posts on this site address measurement of pH in organic media.

$egin hline ext hline egin T (°C) &0.0416 & 0.0891 & 0.2068 & 0.4771 hline -5.0000 & 4.2660 & 4.5700 & 5.1120 & 5.5270 0.0000 & 4.2490 & 4.5440 & 5.0760 & 5.5000 10.0000 & 4.2350 & 4.5130 & 5.0260 & 5.4690 25.0000 & 4.2360 & 4.5080 & 4.9760 & 5.4720 40.0000 & 4.2600 & 4.5340 & 4.9780 & 5.4930 hline endend$

Biology: Can a low pH denature an enzyme or is a just a high pH?

Yep, pHs above and below the optimum can denature the enzymes for the same reason

Enzymes are sensitive to conditions other than their optimum.
Higher pH = Denature
Lower pH = Denature
Higher temperature = Denature
Lower temperature = Work way too slowly to keep up the reactions in the body.

To answer your question:
Yes, a low pH can also denature an enzyme. It doesn't matter if it is a higher pH than normal or a lower pH than normal if there is a pH change then it will change the shape of the active site and render the enzyme useless.

(Original post by AlexeiLipov)
Enzymes are sensitive to conditions other than their optimum.
Higher pH = Denature
Lower pH = Denature
Higher temperature = Denature
Lower temperature = Work way too slowly to keep up the reactions in the body.

To answer your question:
Yes, a low pH can also denature an enzyme. It doesn't matter if it is a higher pH than normal or a lower pH than normal if there is a pH change then it will change the shape of the active site and render the enzyme useless.

Enzyme Activity Review in 26 Easy Questions

Catalysts are substances that reduce the activation energy of a chemical reaction, facilitating it or making it energetically viable. The catalyst increases the speed of the chemical reaction.

More Bite-Sized Q&As Below

2. What amount of catalyst is consumed in the reaction it catalyzes?

Catalysts are not consumed in the reactions they catalyze.

3. Is there a difference between the initial and the final energy levels in catalyzed and non-catalyzed reactions?

The catalysis does not alter the state of the energy of the reagents and products of a chemical reaction. Only the energy necessary for the reaction to occur, that is, the activation energy, is altered.

4. What are enzymes? What is the importance of enzymes for living beings?

Enzymes are proteins that are catalysts of chemical reactions. Chemistry shows us that catalysts are non-consumable substances that reduce the activation energy necessary for a chemical reaction to occur.

Enzymes are highly specific to the reactions they catalyze. They are of vital importance for life because most of the chemical reactions in cells and tissues are catalyzed by enzymes. Without enzyme action, those reactions would not occur or would not happen with the required speed for the biological processes in which they are involved.

The Enzyme-Substrate Complex

5. What are substrates of enzymatic reactions?

Substrates are reagent molecules upon which enzymes act.

Enzymes have spatial binding sites to attach to their substrate. These sites are called the activation centers of the enzyme. Substrates bind to these centers, forming the enzyme-substrate complex.

6. What are the main theoretical models that try to explain the formation of the enzyme-substrate complex?

There are two main models that explain the formation of the enzyme-substrate complex: the lock and key model and the induced fit model.

In the lock and key model, the enzyme has a region with a specific spatial conformation for the binding of the substrate. In the induced fit model, the binding of the substrate induces a change in the spatial configuration of the enzyme to make the substrate fit.

7. How does the formation of the enzyme-substrate complex explain the reduction in the activation energy of chemical reactions?

The enzyme possibly works as like a test tube within which reagents meet to form products. Enzymes facilitate this meeting, making it easier for collisions between reagents to occur and, as a result, the activation energy of the chemical reaction is reduced. This is one possible hypothesis.

8. On what structural level of the enzyme (primary, secondary, tertiary or quaternary) does the enzyme-substrate interaction depend?

The substrate binds to the enzyme at the activation centers. These are specific three-dimensional sites and therefore they depend on the protein's tertiary and quaternary structures. The primary and secondary structures, however, condition the other structures, and consequently are equally important.

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Specificity of Enzymatic Action

9. What is the activation center of an enzyme? Is it the key or the lock in the lock and key model?

The activation center is a region of the enzyme produced by its spatial conformation to which the substrate binds. In the lock and key model, the activation center is the lock and the substrate is the key.

10. Why enzyme action is considered highly specific?

Enzyme action is highly specific because only the specific substrates of an enzyme bind to the activation center of that enzyme. Each enzyme generally catalyzes only one specific chemical reaction.

Factors that Change Enzyme Activity

11. What happens to the functionality of a denatured enzyme? How can that result be explained with the help of the lock and key model?

According to the lock and key, enzyme functionality depends entirely on the integrity of the activation center, a molecular region with specific spatial characteristics. After denaturation, the spatial conformation of the protein is modified, the activation center is destroyed and the enzyme loses its catalytic activity.

12. What are the main factors that alter the speed of enzymatic reactions?

The main factors that change the speed of enzymatic reactions are temperature, pH and substrate concentration (quantity).

13. How does substrate concentration affect the speed of enzymatic reactions?

Initially, as substrate concentration increases, the speed of the reaction increases. This happens because free activation centers of the enzyme bind to free substrates. Once all the activation centers of the available enzymes are bound to their substrates, new increases in the substrate concentration will have no effect on the speed of the reaction.

14. How does temperature affect the action of enzymes on their substrates?

There are defined temperature ranges under which enzymes operate and there is a specific temperature level (optimum temperature) in which enzymes have maximum efficiency. Therefore, temperature variations affect enzyme activity and the speed of the reactions they catalyze.

In addition, because they are proteins, enzymes can be denatured under extreme temperatures.

15. Concerning enzymatic reactions, how different are the curves of the graph of the variation in the speed of a reaction as function of substrate concentration and the graph of the variation in the speed of a reaction as function of temperature?

The curve of the variation in speed of the enzymatic reaction as a function of increasing substrate concentration increases in a curve formation until approaching the point where it stabilizes due to the saturation of the activationꃎnters of the enzymes.

The curve of the variation in the speed of the enzymatic reaction as a function of increasing temperature initially increases and then reaches a peak (the optimum temperature), after which it decreases to zero at the point in which the enzymes are rendered inactive by denaturation.

16. What is the relationship between  the cooling of organs and tissues for medical transplants and the effect of temperature on enzymatic reactions?

Molecular degradation during the decomposition of organs and tissues is catalyzed by enzymes. The cooling to adequate temperatures of some organs and tissues destined for transplantation reduces that enzyme activity and thus decreases the natural decomposition process. By the same rationale, the cooling reduces the metabolic work of cells and prevents the breakdown of their own structures to obtain energy. A subsequent increase in temperature reverts the denaturation of enzymes, allowing the organs and tissues also preserved by other specific techniques to be grafted into the receptors.

17. Does pH affect enzyme activity?

The concentration of hydrogen ions in a solution affects enzyme activity. Each enzyme has a maximum efficiency in an optimum pH.

Since pH is one of the factors in the denaturation of proteins, if an enzyme is subject to a pH level under which it is denatured, there will be no enzymatic activity.

18. Do enzymes act better under acidic or alkaline pHs?

Most enzymes act under pHs between 6 and 8, a range that corresponds to the general acidic level of cells and blood. There are enzymes, however, that act only under very acid or very alkaline pH. Therefore, enzyme activity depends on pH range.

In the stomach, for example, gastric juice has a very low pH, around 2. Nonetheless, the enzyme pepsin acts to intensively digest proteins. In the duodenum, pancreatic secretions increase the pH of the intestinal juice to allow other digestive enzymes, such as trypsin, to act.

19. Since pepsin is a gastric enzyme, does it have an acidic or alkaline optimum pH? What happens to pepsin when it enters the duodenum?

Pepsin acts within the stomach so its optimum pH is around 2, an acidic pH. When the enzyme enters the duodenum, it comes in contact with a higher pH and its enzyme activity comes to and end.


20. What are enzyme cofactors?

Some enzymes need other associated molecules to work. These molecules are called enzyme cofactors and they can be organic ions like mineral salts, or organic molecules, to give some examples.

Inactive enzymes which are not bound to their cofactors are called apoenzymes. Active enzymes bound to their cofactors are called holoenzymes.

21. What is the relationship between vitamins and enzyme cofactors?

Many vitamins are enzyme cofactors that cannot be synthesized by the body and, as a result, must be obtained from the diet.

Enzyme Inhibitors, Allosterism and Zymogens

22. In a enzymatic reaction, what is the effect of a substance with the same spatial conformation as the enzyme substrate? How is this type of substance recognized?

Substances that “simulate” substrates can bind to the activation center of enzymes, thus blocking the true substrates from binding to these enzymes and paralyzing the enzymatic reaction. These “fake substrates” are called enzyme inhibitors.

The binding of enzyme inhibitors to enzymes can be reversible or irreversible.

Many medical drugs, including some antibiotics, antivirals, antineoplastics, antihypertensives and even sildenafil (trade name Viagra), are enzyme inhibitors that block enzyme activity.

23. What is the mechanism of action of the antibiotic penicillin?

Penicillin, discovered by the Scottish doctor Alexander Fleming in 1928, is a drug that inhibits the enzymes necessary for the synthesis of peptidoglycans, a component of the bacterial cell wall. Through this, the inhibition the bacterial population stops growing because there is no new cell wall formation.

Fleming won the Nobel Prize in medicine for the discovery of penicillin.

24. What is the mechanism of action of the antiretroviral drugs called protease inhibitors which are used against HIV infection?

Protease inhibitors are some of the antiretroviral drugs used to treat HIV infection. Protease is an enzyme necessary for the construction of the  human immunodeficiency virus (HIV)ꂯter the synthesis of its proteins within the host cell. The protease inhibitor binds to the activation center of the enzyme blocking the formation of the enzyme-substrate complex and enzyme activity, thus stopping viral replication.

25. What are allosteric enzymes?

Allosteric enzymes are enzymes with more than one activation center and to which other substances, called allosteric regulators, bind.

Allosteric regulators can be allosteric inhibitors or allosteric activators. The interaction between an allosteric enzyme and an allosteric inhibitor prohibits the binding of the substrate to the enzyme. The interaction between an allosteric enzyme and an allosteric activator allows the binding of the substrate to the enzyme and sometimes increases the affinity of the enzyme for the substrate. This regulatory phenomenon of enzyme activity is called allosterism.

26. What are zymogens?

Zymogens, or proenzymes, are enzymes secreted in inactive form. Under certain conditions, a zymogen changes into the active form of the enzyme. In general, zymogen secretions happen because enzyme activity can harm secretory tissue.

For example, the pepsinogen secreted by the stomach becomes active under an acidic pH, turning into the enzyme pepsin. Other well-known zymogens are trypsinogen and chymotrypsinogen, enzymes that are secreted by the exocrine pancreas and which become trypsin and chymotrypsin respectively.

Why does high pH result in the denaturation of DNA? - Biology


Principles of Polyacrylamide Gel Electrophoresis (PAGE)

Powerful electrophoretic techniques have been developed to separate macromolecules on the basis of molecular weight. The mobility of a molecule in an electric field is inversely proportional to molecular friction which is the result of its molecular size and shape, and directly proportional to the voltage and the charge of the molecule. Proteins could be resolved electrophoretically in a semi-solid matrix strictly on the basis of molecular weight if, at a set voltage, a way could be found to charge these molecules to the same degree and to the same sign. Under these conditions, the mobility of the molecules would be simply inversely proportional to their size.

It is precisely this idea which is exploited in PAGE to separate polypeptides according to their molecular weights. In PAGE, proteins charged negatively by the binding of the anionic detergent SDS (sodium dodecyl sulfate) separate within a matrix of polyacrylamide gel in an electric field according to their molecular weights.
Polyacrylamide is formed by the polymerization of the monomer molecule-acrylamide crosslinked by N,N'-methylene-bis-acrylamide (abbreviated BIS). Free radicals generated by ammonium persulfate (APS) and a catalyst acting as an oxygen scavenger (-N,N,N',N'-tetramethylethylene diamine [TEMED]) are required to start the polymerization since acrylamide and BIS are nonreactive by themselves or when mixed together.

The distinct advantage of acrylamide gel systems is that the initial concentrations of acrylamide and BIS control the hardness and degree of crosslinking of the gel. The hardness of a gel in turn controls the friction that macromolecules experience as they move through the gel in an electric field, and therefore affects the resolution of the components to be separated. Hard gels (12-20% acrylamide) retard the migration of large molecules more than they do small ones. In certain cases, high concentration acrylamide gels are so tight that they exclude large molecules from entering the gel but allow the migration and resolution of low molecular weight components of a complex mixture. Alternatively, in a loose gel (4-8% acrylamide), high molecular weight molecules migrate much farther down the gel and, in some instances, can move right out of the matrix.

SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Sodium dodecyl sulfate (SDS or sodium lauryl sulfate) is an anionic detergent which denatures proteins molecules without breaking peptide bonds. It binds strongly to all proteins and creates a very high and constant charge:mass ratio for all denatured proteins. After treatment with SDS, irrespective of their native charges, all proteins acquire a high negative charge.

Denaturation of proteins is performed by heating them in a buffer containing a soluble thiol reducing agent (e.g. 2-mercaptoethanol dithiothreitol) and SDS. Mercaptoethanol reduces all disulfide bonds of cysteine residues to free sulfhydryl groups, and heating in SDS disrupts all intra- and intermolecular protein interactions. This treatment yields individual polypeptide chains which carry an excess negative charge induced by the binding of the detergent, and an identical charge:mass ratio. Thereafter, the denatured proteins can be resolved electrophoretically strictly on the basis of size in a buffered polyacrylamide gel which contains SDS and thiol reducing agents.

SDS-PAGE gel systems are exceedingly useful in analyzing and resolving complex protein mixtures. Many applications and modifications of this technique are relevant to modern experimental biologists. Some are mentioned below. They are employed to monitor enzyme purification, to determine the subunit composition of oligomeric proteins, to characterize the protein components of subcellular organelles and membranes, and to assign specific proteins to specific genes by comparing protein extracts of wild-type organisms and suppressible mutants. In addition, the mobility of polypeptides in SDS-PAGE gel systems is proportional to the inverse of the log of their molecular weights. This property makes it possible to measure the molecular weight of an unknown protein with an accuracy of +/- 5%, quickly, cheaply and reproducibly.

Discontinuous SDS Polyacrylamide Gel Electrophoresis

Disc gels are constructed with two different acrylamide gels, one on top of the other. The upper or stacking gel contains 4-5% acrylamide (a very loose gel) weakly buffered at pH 9.0. The lower resolving gel (often called the running gel), contains a higher acrylamide concentration, or a gradient of acrylamide, strongly buffered at pH 9.0. Both gels can be cast as tubes in glass or plastic cylinders (tube gels), or as thin slabs within glass plates, an arrangement which improves resolution considerably, and which makes it possible to analyze and compare many protein samples at once, and on the same gel (slab gels). Today, you will be constructing and running slab gels.

The ionic strength discontinuity between the loose stacking gel and the hard running gel leads to a voltage discontinuity as current is applied. The goal of these gels is to maximize resolution of protein molecules by reducing and concentrating the sample to an ultrathin zone (1-100 nm) at the stacking gel:running gel boundary. The protein sample is applied in a well within the stacking gel as a rather long liquid column (0.2-0.5 cm) depending on the amount and the thickness of the gel or tube. The protein sample contains glycerol or sucrose so that it can be overlaid with a running buffer. This buffer is called the running buffer, and the arrangement is such that the top and bottom of the gel are in running buffer to make a circuit.

As current is applied, the proteins start to migrate downward through the stacking gel toward the positive pole, since they are negatively charged by the bound SDS. Since the stacking gel is very loose, low and average molecular weight proteins are not impeded in their migration and move much more quickly than in the running gel. In addition, the lower ionic strength of the stacking gel (weak buffer) creates a high electrical resistance, (i.e., a high electric field V/cm) to make proteins move faster than in the running gel (high ionic strength, lower resistance, hence lower electric field, V/cm). Remember that applied voltage results in current flow in the gel through the migration of ions. Hence low ionic strength means high resistance because fewer ions are present to dissipate the voltage and the electric field (V/cm) is increased causing the highly polyanionic proteins to migrate rapidly.

The rapid migration of proteins through the stacking gel causes them to accumulate and stack as a very thin zone at the stacking gel/running gel boundary, and most importantly, since the 4-5% stacking gel affects the mobility of the large components only slightly, the stack is arranged in order of mobility of the proteins in the mixture. This stacking effect results in superior resolution within the running gel, where polypeptides enter and migrate much more slowly, according to their size and shape.

In all gel systems, a tracking dye (usually Bromophenol blue) is introduced with the protein sample to determine the time at which the operation should be stopped. Bromophenol blue is a small molecule which travels essentially unimpeded just behind the ion front moving down toward the bottom of the gel. Few protein molecules travel ahead of this tracking dye. When the dye front reaches the bottom of the running gel, the current is turned off to make sure that proteins do not electrophorese out of the gel into the buffer tank.

Visualizing the Proteins

Gels are removed from tubes or from the glass plates and stained with a dye, Coomassie Brilliant Blue. Coomassie blue binds strongly to all proteins. Unbound dye is removed by extensive washing of the gel. Blue protein bands can thereafter be located and quantified since the amount of bound dye is proportional to protein content. Stained gels can be dried and preserved, photographed or scanned with a recording densitometer to measure the intensity of the color in each protein band. Alternatively, if the proteins are radioactive, the protein bands can be detected by autoradiography, a technique that is widely used in modern cell and molecular biology. When gels are prepared as thin slabs to maximize resolution as you will do today, the slabs of acrylamide are removed from the support glass plates and dried on filter paper. A piece of X-ray film is placed and clamped tight over the dried slab in a light-proof box. The X-ray film is exposed by the radioactivity in the protein bands and, after developing, dark spots or bands can be seen on the film. These dark bands can in turn be quantified since their intensity is proportional to the amount of radioactivity and hence to protein content.

ventilation is moving air into and out of lungs/inhalation and exhalation
involves (respiratory) muscle activity
gas exchange involves movement of carbon dioxide and oxygen
between alveoli and blood (in capillaries) / between blood (in capillaries) and cells
cell respiration is the release of energy from organic molecules/glucose
(aerobic) cell respiration occurs in mitochondria
To award [4 max] responses must address ventilation, gas exchange and cell respiration.

during glycolysis glucose is partially oxidized in the cytoplasm
(small amount/yield of) ATP produced
(two) pyruvate formed by glycolysis
pyruvate absorbed into/broken down in the mitochondrion
requires oxygen
carbon dioxide is produced
water is produced
large amount/yield of energy/ATP molecules (per glucose molecule)

collisions between enzyme/active site and substrate
enzyme activity increases as temperature rises
more frequent collisions at higher temperatures
each enzyme has an optimum temperature / enzymes have optimal temperatures
high temperatures (above optimum) denature enzymes
each enzyme has an optimum pH / enzymes have optimal pHs
increase or decrease from optimum pH decreases rate of reaction/activity
extreme pH alters/denatures the tertiary/3D protein/enzyme structure
increasing substrate concentration increases the rate of reaction
higher substrate concentration increases chance of collision
until plateau
when all active sites are busy
Accept clearly annotated graph.

Examiners report

  • 17N.1.SL.TZ0.09: Three flasks were prepared for an analysis of the activity of amylase. At time zero, each of.
  • 17M.3.SL.TZ1.1c: Outline the effect of temperature on the activity of urease enzyme.
  • 17M.3.SL.TZ1.1b: One result in this experiment can be classified as an outlier as its value is very distant.
  • 17M.3.SL.TZ1.1a: Outline what the standard deviations reveal about the data from this experiment.
  • 17M.2.HL.TZ1.1f.ii: Suggest a reason for the greater expression of the gene for the urea transporter after an.
  • 17M.2.SL.TZ1.1c: Estimate how much smaller drilled oysters raised in seawater at a high CO2 concentration were.
  • 17M.1.SL.TZ2.27: The bacterium Neisseria gonorrhoeae causes infections related to the human reproductive.
  • 17M.1.SL.TZ1.7: In an experiment the effect of changing pH on an enzymatic reaction is tested. Which could be.
  • 17M.1.SL.TZ1.21: Cladograms can be created by comparing DNA or protein sequences. The cladogram on the left is.
  • 17M.1.SL.TZ1.10: The graph shows the effect of increasing the substrate concentration on the rate of an.
  • 16N.3.HL.TZ0.1c: Explain what would happen to fish protein hydrolysis if no alkali were added to the reaction.
  • 16N.3.HL.TZ0.1b: Sketch on the graph the curve expected if the hydrolysis were performed using papain 0.5 %.
  • 16N.3.HL.TZ0.1a: State the effect of enzyme concentration on the hydrolysis of proteins.
  • 16N.3.SL.TZ0.2c: Explain the importance of having equal quantities of the enzyme at the start of the experiment.
  • 16N.3.SL.TZ0.2b: Suggest one method that could have been used to keep the tubes at a constant temperature.
  • 16N.3.SL.TZ0.2a: Suggest one reason for differences between the cereal grains, in the percentage of starch.
  • 16M.2.HL.TZ0.5a: Outline the action of enzymes.
  • 16N.1.HL.TZ0.9: It is possible to attach β-galactosidase to alginate beads for use in the production of.
  • 16N.1.SL.TZ0.9: A fever in a normally healthy adult during an illness is not usually a problem and can be.
  • 15M.1.HL.TZ1.9: Why does exposure to high temperatures cause an enzyme to lose its biological properties?A.
  • 15M.1.SL.TZ2.10: How can the activity of a human amylase enzyme be increased during a laboratory experiment?A.
  • 15M.2.SL.TZ1.2c: Milk contains lactose which some people can digest but some cannot. Explain the production.
  • 15M.1.HL.TZ2.11: Which is the activation energy of a reaction when it is catalysed by an enzyme?
  • 15M.2.HL.TZ1.2a (ii): Explain the production of lactose-free milk.
  • 15N.1.HL.TZ0.8: What is decreased when lactase is added to milk? A. Sweetness B. Disaccharides C.
  • 13M.2.HL.TZ2.4a: Define the active site of an enzyme.
  • 13M.2.HL.TZ2.4b: Explain how the active site promotes enzyme–substrate specificity.
  • 13M.2.HL.TZ2.4c: Outline possible effects of acids on enzyme activity.
  • 13M.1.HL.TZ1.8: Which graph shows the effect of increasing substrate concentration on enzyme activity?
  • 13M.1.HL.TZ1.9: What is produced when the enzyme lactase is added to milk? A. Glucose and galactoseB.
  • 13M.1.HL.TZ2.10: For what purpose is the enzyme lactase useful? A. Production of lactose-free milk so that.
  • 13N.1.SL.TZ0.9: In enzyme experiments, the rate of enzyme activity often gradually decreases. What is most.
  • 13M.2.SL.TZ2.5c: Explain the effect of changes of pH, substrate concentration and temperature on enzyme activity.
  • 13N.2.SL.TZ0.7b: Some proteins in membranes act as enzymes. Outline enzyme-substrate specificity.
  • 13M.1.SL.TZ2.10: What contributes to the structure of an enzyme? A. Sequence of bases linked by hydrogen.
  • 13M.3.SL.TZ2.8b: Explain the role of enzymes in metabolic pathways.
  • 11M.2.HL.TZ2.5a: Outline the effect of temperature and substrate concentration on the activity of enzymes.
  • 11M.1.SL.TZ1.8: The graph below shows the effect of substrate concentration on enzyme activity. What.
  • 11M.1.SL.TZ1.11: What is denaturation? A. A structural change of a protein that results in the loss of its.
  • 11M.1.SL.TZ2.30: Which variable has the least effect on enzyme activity? A. Temperature B. Light intensity.
  • 12M.1.HL.TZ1.9: Which graph shows the effect of increasing the substrate concentration on enzyme activity?
  • 12M.1.SL.TZ2.10: How does an increase in temperature affect enzyme activity?
  • 09M.2.HL.TZ1.6c: Outline how enzymes catalyse reactions.
  • 09M.2.SL.TZ1.3b: Explain enzyme-substrate specificity.
  • 09M.2.SL.TZ1.3a: Define active site.
  • 09M.2.SL.TZ2.3b: Explain the effects of pH on enzyme catalysed reactions.
  • 09M.1.HL.TZ2.34: Which of the following statements is true about enzymes? A. They are used up in the.
  • 09M.1.SL.TZ1.11: What happens as an enzyme becomes denatured? A. The enzyme works faster. B. The enzyme works.
  • 10M.2.HL.TZ1.2d: Simple laboratory experiments show that when the enzyme lactase is mixed with lactose, the.
  • 10M.2.SL.TZ1.2d: Simple laboratory experiments show that when the enzyme lactase is mixed with lactose, the.
  • 10M.2.SL.TZ2.5a: Outline the role of hydrolysis in the relationships between monosaccharides, disaccharides.
  • 10M.2.SL.TZ2.5b: Describe the use of biotechnology in the production of lactose-free milk.
  • 10M.2.SL.TZ2.5c: Explain the importance of enzymes to human digestion.
  • 11N.2.HL.TZ0.5 b: Many people cannot digest lactose and benefit from a diet containing no lactose. Outline the.
  • 11N.2.SL.TZ0.7c: Respiration and other processes in cells involve enzymes. Explain the factors that can affect.
  • 12N.2.SL.TZ0.5b: Metabolic reactions are catalysed by enzymes. Explain how enzymes catalyse reactions and how.


This work was supported in part by grants (No. 30230100 and No. 30670420) from the Natural Science Foundation of China.

We are very grateful to Professor J. F. Wang from the Institute of Biophysics, Chinese Academy of Sciences, and Professor L. Huang from the Institute of Microbiology, Chinese Academy of Sciences, for kindly providing the Ssh10b gene. We also express our sincere thanks to Dr. Sarah Perrett of the Institute of Biophysics, Chinese Academy of Sciences, for comments on this manuscript.


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