during a laboratory experiment you discover that an enzyme catalyzed

Unit 2: Chapter 8 Flashcards

• 1) Which term most precisely describes the cellular process of breaking down large molecules into smaller ones?
• A) catalysis
• B) metabolism
• C) anabolism
• D) dehydration
• E) catabolism
• 2) Which of the following is (are) true for anabolic pathways?
• A) They do not depend on enzymes.
• B) They are usually highly spontaneous chemical reactions.
• C) They consume energy to build up polymers from monomers.
• D) They release energy as they degrade polymers to monomers.
• E) They consume energy to decrease the entropy of the organism and its environment.
• 3) Which of the following is a statement of the first law of thermodynamics?
• A) Energy cannot be created or destroyed.
• B) The entropy of the universe is decreasing.
• C) The entropy of the universe is constant.
• D) Kinetic energy is stored energy that results from the specific arrangement of matter.
• E) Energy cannot be transferred or transformed.
• A) The energy content of an organism is constant.
• B) The organism ultimately must obtain all of the necessary energy for life from its environment.
• C) The entropy of an organism decreases with time as the organism grows in complexity.
• D) Organisms grow by converting energy into organic matter.
• E) Life does not obey the first law of thermodynamics.
• A) Living organisms do not obey the second law of thermodynamics, which states that entropy must increase with time.
• B) Life obeys the second law of thermodynamics because the decrease in entropy as the organism grows is exactly balanced by an increase in the entropy of the universe.
• C) Living organisms do not follow the laws of thermodynamics.
• D) As a consequence of growing, organisms cause a greater increase in entropy in their environment than the decrease in entropy associated with their growth.
• E) Living organisms are able to transform energy into entropy.
• Whenever energy is transformed, there is always an increase in the
• A) free energy of the system.
• B) free energy of the universe.
• C) entropy of the system.
• D) entropy of the universe.
• E) enthalpy of the universe.
• A) If the entropy of a system increases, there must be a corresponding decrease in the entropy of the universe.
• B) If there is an increase in the energy of a system, there must be a corresponding decrease in the energy of the rest of the universe.
• C) Every energy transfer requires activation energy from the environment.
• D) Every chemical reaction must increase the total entropy of the universe.
• E) Energy can be transferred or transformed, but it cannot be created or destroyed.
• A) Conversion of energy from one form to another is always accompanied by some gain of free energy.
• B) Heat represents a form of energy that can be used by most organisms to do work.
• C) Without an input of energy, organisms would tend toward decreasing entropy.
• D) Cells require a constant input of energy to maintain their high level of organization.
• E) Every energy transformation by a cell decreases the entropy of the universe.
• 9) Which of the following types of reactions would decrease the entropy within a cell?
• A) anabolic reactions
• B) hydrolysis
• C) respiration
• D) digestion
• E) catabolic reactions
• A) has occurred in accordance with the laws of thermodynamics.
• B) has caused an increase in the entropy of the planet.
• C) has been made possible by expending Earthʹs energy resources.
• D) has occurred in accordance with the laws of thermodynamics, by expending Earthʹs energy resources and causing an increase in the entropy of the planet.
• E) violates the laws of thermodynamics because Earth is a closed system.
• 11) Which of the following is an example of potential rather than kinetic energy?
• A) the muscle contractions of a person mowing grass
• B) water rushing over Niagara Falls
• C) light flashes emitted by a firefly
• D) a molecule of glucose
• E) the flight of an insect foraging for food
• 12) Which of the following is the smallest closed system?
• B) an organism
• C) an ecosystem
• E) the universe
• 13) Which of the following is true of metabolism in its entirety in all organisms?
• A) Metabolism depends on a constant supply of energy from food.
• B) Metabolism depends on an organismʹs adequate hydration.
• C) Metabolism uses all of an organismʹs resources.
• D) Metabolism consists of all the energy transformation reactions in an organism.
• E) Metabolism manages the increase of entropy in an organism.
• 14) The mathematical expression for the change in free energy of a system is Δ G =ΔH - TΔS. Which of the following is (are) correct?
• A) ΔS is the change in enthalpy, a measure of randomness.
• B) ΔH is the change in entropy, the energy available to do work.
• C) ΔG is the change in free energy.
• D) T is the temperature in degrees Celsius.
• 15) A system at chemical equilibrium
• A) consumes energy at a steady rate.
• B) releases energy at a steady rate.
• C) consumes or releases energy, depending on whether it is exergonic or endergonic.
• D) has zero kinetic energy.
• E) can do no work.
• A) The products have more total energy than the reactants.
• B) The reaction proceeds with a net release of free energy.
• C) The reaction goes only in a forward direction: all reactants will be converted to products, but no products will be converted to reactants.
• D) A net input of energy from the surroundings is required for the reactions to proceed.
• E) The reactions are rapid.
• A) a reaction in which the free energy at equilibrium is higher than the energy content at any point away from equilibrium
• B) a chemical reaction in which the entropy change in the reaction is just balanced by an opposite entropy change in the cellʹs surroundings
• C) an endergonic reaction in an active metabolic pathway where the energy for that reaction is supplied only by heat from the environment
• D) a chemical reaction in which both the reactants and products are not being produced or used in any active metabolic pathway
• E) no possibility of having chemical equilibrium in any living cell
• 18) Which of the following shows the correct changes in thermodynamic properties for a chemical reaction in which amino acids are linked to form a protein?
• A) +ΔH, +ΔS, +ΔG
• B) +ΔH, -ΔS, -ΔG
• C) +ΔH, -ΔS, +ΔG
• D) -ΔH, -ΔS, +ΔG
• E) -ΔH, +ΔS, +ΔG
• 19) When glucose monomers are joined together by glycosidic linkages to form a cellulose polymer, the changes in free energy, total energy, and entropy are as follows:
• A) +ΔG, +ΔH, +ΔS.
• B) +ΔG, +ΔH, -ΔS.
• C) +ΔG, -ΔH, -ΔS.
• D) -ΔG, +ΔH, +ΔS. E) -ΔG, -ΔH, -ΔS.
• 20) A chemical reaction that has a positive ΔG is correctly described as
• A) endergonic.
• B) endothermic.
• C) enthalpic.
• D) spontaneous.
• E) exothermic.
• 21) Which of the following best describes enthalpy (H)?
• A) the total kinetic energy of a system
• B) the heat content of a chemical system
• C) the systemʹs entropy
• D) the cellʹs energy equilibrium
• E) the condition of a cell that is not able to react
• 22) For the hydrolysis of ATP to ADP + i, the free energy change is -7.3 kcal/mol under standard conditions (1 M concentration of both reactants and products). In the cellular environment, however, the free energy change is about -13 kcal/mol. What can we conclude about the free energy change for the formation of ATP from ADP and under cellular conditions?
• A) It is +7.3 kcal/mol.
• B) It is less than +7.3 kcal/mol.
• C) It is about +13 kcal/mol.
• D) It is greater than +13 kcal/mol.
• E) The information given is insufficient to deduce the free energy change.

23) Why is ATP an important molecule in metabolism?

• A) Its hydrolysis provides an input of free energy for exergonic reactions.
• B) It provides energy coupling between exergonic and endergonic reactions.
• C) Its terminal phosphate group contains a strong covalent bond that, when hydrolyzed, releases free energy.
• D) Its terminal phosphate bond has higher energy than the other two.
• E) It is one of the four building blocks for DNA synthesis.

24) When 10,000 molecules of ATP are hydrolyzed to ADP and i in a test tube, about twice as much heat is liberated as when a cell hydrolyzes the same amount of ATP. Which of the following is the best explanation for this observation?

• A) Cells are open systems, but a test tube is a closed system.
• B) Cells are less efficient at heat production than nonliving systems.
• C) The hydrolysis of ATP in a cell produces different chemical products than does the reaction in a test tube.
• D) The reaction in cells must be catalyzed by enzymes, but the reaction in a test tube does not need enzymes.
• E) Reactant and product concentrations in the test tube are different from those in the cell.
• 25) Which of the following is most similar in structure to ATP?
• A) a pentose sugar
• B) a DNA nucleotide
• C) an RNA nucleotide
• D) an amino acid with three phosphate groups attached
• E) a phospholipid
• 26) Which of the following statements is true concerning catabolic pathways?
• A) They combine molecules into more energy-rich molecules.
• B) They supply energy, primarily in the form of ATP, for the cellʹs work.
• C) They are endergonic.
• D) They are spontaneous and do not need enzyme catalysis.
• E) They build up complex molecules such as protein from simpler compounds.
• 27) When chemical, transport, or mechanical work is done by an organism, what happens to the heat generated?
• A) It is used to power yet more cellular work.
• B) It is used to store energy as more ATP.
• C) It is used to generate ADP from nucleotide precursors.
• D) It is lost to the environment.
• E) It is transported to specific organs such as the brain.
• 28) When ATP releases some energy, it also releases inorganic phosphate. What purpose does this serve (if any) in the cell?
• A) The phosphate is released as an excretory waste.
• B) The phosphate can only be used to regenerate more ATP.
• C) The phosphate can be added to water and excreted as a liquid.
• D) The phosphate may be incorporated into any molecule that contains phosphate.
• E) It enters the nucleus to affect gene expression.
• A) ATPase activity must be powering an inflow of calcium from the outside of the cell into the SR.
• B) ATPase activity must be transferring i to the SR to enable this to occur.
• C) ATPase activity must be pumping calcium from the cytosol to the SR against the concentration gradient.
• D) ATPase activity must be opening a channel for the calcium ions to diffuse back into the SR along the concentration gradient.
• E) ATPase activity must be routing calcium ions from the SR to the cytosol, and then to the cellʹs environment.
• 30) What is the difference (if any) between the structure of ATP and the structure of the precursor of the A nucleotide in RNA?
• A) The sugar molecule is different.
• B) The nitrogen-containing base is different.
• C) The number of phosphates is three instead of one.
• D) The number of phosphates is three instead of two.
• E) There is no difference.
• A) The reaction is faster than the same reaction in the absence of the enzyme.
• B) The free energy change of the reaction is opposite from the reaction that occurs in the absence of the enzyme.
• C) The reaction always goes in the direction toward chemical equilibrium.
• D) Enzyme-catalyzed reactions require energy to activate the enzyme.
• E) Enzyme-catalyzed reactions release more free energy than noncatalyzed reactions.
• 32) Reactants capable of interacting to form products in a chemical reaction must first overcome a thermodynamic barrier known as the reactionʹs
• A) entropy.
• B) activation energy.
• C) endothermic level.
• D) equilibrium point.
• E) free-energy content.
• 33) A solution of starch at room temperature does not readily decompose to form a solution of simple sugars because
• A) the starch solution has less free energy than the sugar solution.
• B) the hydrolysis of starch to sugar is endergonic.
• C) the activation energy barrier for this reaction cannot be surmounted.
• D) starch cannot be hydrolyzed in the presence of so much water.
• E) starch hydrolysis is nonspontaneous.
• 34) Which of the following statements regarding enzymes is true?
• A) Enzymes increase the rate of a reaction by making the reaction more exergonic.
• B) Enzymes increase the rate of a reaction by lowering the activation energy barrier.
• C) Enzymes increase the rate of a reaction by reducing the rate of reverse reactions.
• D) Enzymes change the equilibrium point of the reactions they catalyze.
• E) Enzymes make the rate of a reaction independent of substrate concentrations.
• 35) During a laboratory experiment, you discover that an enzyme-catalyzed reaction has a ∆G of -20 kcal/mol. If you double the amount of enzyme in the reaction, what will be the ∆G for the new reaction?
• A) -40 kcal/mol
• B) -20 kcal/mol
• C) 0 kcal/mol
• D) +20 kcal/mol
• E) +40 kcal/mol
• 36) The active site of an enzyme is the region that
• A) binds allosteric regulators of the enzyme.
• B) is involved in the catalytic reaction of the enzyme.
• C) binds noncompetitive inhibitors of the enzyme.
• D) is inhibited by the presence of a coenzyme or a cofactor.
• 37) According to the induced fit hypothesis of enzyme catalysis, which of the following is correct?
• A) The binding of the substrate depends on the shape of the active site.
• B) Some enzymes change their structure when activators bind to the enzyme.
• C) A competitive inhibitor can outcompete the substrate for the active site.
• D) The binding of the substrate changes the shape of the enzymeʹs active site.
• E) The active site creates a microenvironment ideal for the reaction.
• A) can have no effect on the activity or properties of the enzyme.
• B) will almost always destroy the activity of the enzyme.
• C) will often cause a change in the substrate specificity of the enzyme.
• D) may affect the physicochemical properties of the enzyme such as its optimal temperature and pH.
• E) may, in rare cases, cause the enzyme to run reactions in reverse.
• 39) Increasing the substrate concentration in an enzymatic reaction could overcome which of the following?
• A) denaturization of the enzyme
• B) allosteric inhibition
• C) competitive inhibition
• D) saturation of the enzyme activity
• E) insufficient cofactors
• A) Nonprotein cofactors alter the substrate specificity of enzymes.
• B) Enzyme function is increased if the 3-D structure or conformation of an enzyme is altered.
• C) Enzyme function is independent of physical and chemical environmental factors such as pH and temperature.
• D) Enzymes increase the rate of chemical reaction by lowering activation energy barriers.
• E) Enzymes increase the rate of chemical reaction by providing activation energy to the substrate.
• 41) Zinc, an essential trace element for most organisms, is present in the active site of the enzyme carboxypeptidase. The zinc most likely functions as a(n)
• A) competitive inhibitor of the enzyme.
• B) noncompetitive inhibitor of the enzyme.
• C) allosteric activator of the enzyme.
• D) cofactor necessary for enzyme activity.
• E) coenzyme derived from a vitamin.
• A) The ATP must first have to attach to the tRNA.
• B) The binding of the first two molecules must cause a 3-D change that opens another active site on the enzyme.
• C) The ATP must be hydrolyzed to allow the amino acid to bind to the synthetase.
• D) The tRNA molecule must have to alter its shape in order to be able to fit into the active site with the other two molecules.
• E) The 3ʹ end of the tRNA must have to be cleaved before it can have an attached amino acid.
• 43) Some of the drugs used to treat HIV patients are competitive inhibitors of the HIV reverse transcriptase enzyme. Unfortunately, the high mutation rate of HIV means that the virus rapidly acquires mutations with amino acid changes that make them resistant to these competitive inhibitors. Where in the reverse transcriptase enzyme would such amino acid changes most likely occur in drug-resistant viruses?
• A) in or near the active site
• B) at an allosteric site
• C) at a cofactor binding site
• D) in regions of the protein that determine packaging into the virus capsid
• E) such mutations could occur anywhere with equal probability
• A) ATP is more abundant near the plasma membrane.
• B) They can more readily encounter and phosphorylate other membrane proteins.
• C) Membrane localization lowers the activation energy of the phosphorylation reaction.
• D) They flip back and forth across the membrane to access target proteins on either side.
• E) They require phospholipids as a cofactor.
• 45) When you have a severe fever, what grave consequence may occur if the fever is not controlled?
• A) destruction of your enzymesʹ primary structure
• B) removal of amine groups from your proteins
• C) change in the tertiary structure of your enzymes
• D) removal of the amino acids in active sites of your enzymes
• E) binding of your enzymes to inappropriate substrates
• 46) How does a noncompetitive inhibitor decrease the rate of an enzyme reaction?
• A) by binding at the active site of the enzyme
• B) by changing the shape of the enzymeʹs active site
• C) by changing the free energy change of the reaction
• D) by acting as a coenzyme for the reaction
• E) by decreasing the activation energy of the reaction
• 47) The mechanism in which the end product of a metabolic pathway inhibits an earlier step in the pathway is most precisely described as
• A) metabolic inhibition.
• B) feedback inhibition.
• C) allosteric inhibition.
• D) noncooperative inhibition.
• E) reversible inhibition.
• A) A multienzyme complex contains all the enzymes of a metabolic pathway.
• B) A product of a pathway serves as a competitive inhibitor of an early enzyme in the pathway.
• C) A substrate molecule bound to an active site of one subunit promotes substrate binding to the active site of other subunits.
• D) Several substrate molecules can be catalyzed by the same enzyme.
• E) A substrate binds to an active site and inhibits cooperation between enzymes in a pathway.
• 49) Allosteric enzyme regulation is usually associated with
• A) lack of cooperativity.
• C) activating activity.
• D) an enzyme with more than one subunit.
• E) the need for cofactors.
• 50 ) Which of the following is an example of cooperativity?
• A) the binding of an end product of a metabolic pathway to the first enzyme that acts in the pathway
• B) one enzyme in a metabolic pathway passing its product to act as a substrate for the next enzyme in the pathway
• C) a molecule binding at one unit of a tetramer, allowing faster binding at each of the other three
• D) the effect of increasing temperature on the rate of an enzymatic reaction
• E) binding of an ATP molecule along with one of the substrate molecules in an active site
• 51) Protein kinases are enzymes that catalyze phosphorylation of target proteins at specific sites, whereas protein phosphatases catalyze removal of phosphate(s) from phosphorylated proteins. Phosphorylation and dephosphorylation can function as an on-off switch for a proteinʹs activity, most likely through:
• A) the change in a proteinʹs charge leading to a conformational change.
• B) the change in a proteinʹs charge leading to cleavage.
• C) a change in the optimal pH at which a reaction will occur.
• D) a change in the optimal temperature at which a reaction will occur.
• E) the excision of one or more peptides.
• 52) Besides turning enzymes on or off, what other means does a cell use to control enzymatic activity?
• A) cessation of cellular protein synthesis
• B) localization of enzymes into specific organelles or membranes
• C) exporting enzymes out of the cell
• D) connecting enzymes into large aggregates
• E) hydrophobic interactions
• 53) An important group of peripheral membrane proteins are enzymes such as the phospholipases that cleave the head groups of phospholipids. What properties must these enzymes exhibit?
• A) resistance to degradation
• B) independence from cofactor interaction
• C) water solubility
• D) lipid solubility
• E) membrane-spanning domains
• 54) In experimental tests of enzyme evolution, where a gene encoding an enzyme is subjected to multiple cycles of random mutagenesis and selection for altered substrate specificity, the resulting enzyme had multiple amino acid changes associated with altered substrate specificity. Where in the enzyme were these amino acid changes located?
• A) only in the active site
• B) only in the active site or near the active site
• C) in or near the active site and at surface sites away from the active site
• D) only at surface sites away from the active site
• E) only in the hydrophobic interior of the folded protein

55) How might an amino acid change at a site distant from the active site of the enzyme alter the enzymeʹs substrate specificity?

• A) by changing the enzymeʹs stability B) by changing the enzymeʹs location in the cell
• C) by changing the shape of the protein
• D) by changing the enzymeʹs pH optimum
• E) an amino acid change away from the active site cannot alter the enzymeʹs substrate specificity

56) For the enzyme-catalyzed reaction shown in the figure, which of these treatments will cause the greatest increase in the rate of the reaction, if the initial reactant concentration is 1.0 micromolar?

• doubling the activation energy needed
• cooling the reaction by 10°C
• doubling the concentration of the reactants to 2.0 micromolar
• doubling the enzyme concentration
• increasing the concentration of reactants to 10.0 micromolar, while reducing the concentration of enzyme by 1/2

57) In the figure, why does the reaction rate plateau at higher reactant concentrations?

• A) Feedback inhibition by product occurs at high reactant concentrations.
• B) Most enzyme molecules are occupied by substrate at high reactant concentrations.
• C) The reaction nears equilibrium at high reactant concentrations. D) The activation energy for the reaction increases with reactant concentration.
• E) The rate of the reverse reaction increases with reactant concentration.

58) Which curve(s) on the graphs may represent the temperature and pH profiles of an enzyme taken from a bacterium that lives in a mildly alkaline hot springs at temperatures of 70°C or higher?

• A) curves 1 and 5
• B) curves 2 and 4
• C) curves 2 and 5
• D) curves 3 and 4
• E) curves 3 and 5

59) Which temperature and pH profile curves on the graphs were most likely generated from analysis of an enzyme from a human stomach where conditions are strongly acid?

• A) curves 1 and 4
• B) curves 1 and 5
• C) curves 2 and 4
• D) curves 2 and 5
• E) curves 3 and 4

• 60) Which of the following terms best describes the forward reaction in Figure 8.1?
• A) endergonic, ∆G > 0
• B) exergonic, ∆G < 0
• C) endergonic, ∆G < 0
• D) exergonic, ∆G > 0
• E) chemical equilibrium, ∆G = 0

• 61) Which of the following represents the ΔG of the reaction in Figure 8.1?

• 62) Which of the following in Figure 8.1 would be the same in either an enzyme-catalyzed or a noncatalyzed reaction?

• 63) Which of the following represents the activation energy needed for the enzyme-catalyzed reverse reaction, C + D → A + B, in Figure 8.1?

• 64) Which of the following represents the difference between the free-energy content of the reaction and the free-energy content of the products in Figure 8.1?

• 65) Which of the following represents the activation energy required for the enzyme-catalyzed reaction in Figure 8.1?

• 66) Which of the following represents the activation energy required for a noncatalyzed reaction in Figure 8.1?

• 67) Which of the following represents the activation energy needed for the noncatalyzed reverse reaction, C + D → A + B, in Figure 8.1?

• A) The reaction could be coupled to power an endergonic reaction with a ΔG of +6.2 kcal/mol.
• B) The reaction could be coupled to power an exergonic reaction with a ΔG of +8.8 kcal/mol.
• C) The reaction would result in a decrease in entropy (S) and an increase in the total energy content (H) of the system.
• D) The reaction would result in an increase in entropy (S) and a decrease in the total energy content (H) of the system.
• E) The reaction would result in products (C + D) with a greater free-energy content than in the initial reactants (A + B).

69) Which of the following is the most correct interpretation of the figure?

• A) Inorganic phosphate is created from organic phosphate.
• B) Energy from catabolism can be used directly for performing cellular work.
• C) ADP + i are a set of molecules that store energy for catabolism.
• D) ATP is a molecule that acts as an intermediary to store energy for cellular work.
• E) i acts as a shuttle molecule to move energy from ATP to ADP.

70) How do cells use the ATP cycle shown in the figure?

• A) Cells use the cycle to recycle ADP and phosphate.
• B) Cells use the cycle to recycle energy released by ATP hydrolysis.
• C) Cells use the cycle to recycle ADP, phosphate, and the energy released by ATP hydrolysis.
• D) Cells use the cycle to generate or consume water molecules as needed.
• E) Cells use the cycle primarily to generate heat.

Succinate dehydrogenase catalyzes the conversion of succinate to fumarate. The reaction is inhibited by malonic acid, which resembles succinate but cannot be acted upon by succinate dehydrogenase. Increasing the ratio of succinate to malonic acid reduces the inhibitory effect of malonic acid.

• 71) Based on this information, which of the following is correct? A) Succinate dehydrogenase is the enzyme, and fumarate is the substrate.
• B) Succinate dehydrogenase is the enzyme, and malonic acid is the substrate.
• C) Succinate is the substrate, and fumarate is the product. D) Fumarate is the product, and malonic acid is a noncompetitive inhibitor.
• E) Malonic acid is the product, and fumarate is a competitive inhibitor.

72) What is malonic acidʹs role with respect to succinate dehydrogenase?

• A) It is a competitive inhibitor.
• B) It blocks the binding of fumarate.
• C) It is a noncompetitive inhibitor.
• D) It is able to bind to succinate.
• E) It is an allosteric regulator.

A series of enzymes catalyze the reaction X → Y → Z → A. Product A binds to the enzyme that converts X to Y at a position remote from its active site. This binding decreases the activity of the enzyme.

73) What is substance X?

• A) a coenzyme
• B) an allosteric inhibitor
• C) a substrate
• D) an intermediate
• E) the product

74) With respect to the enzyme that converts X to Y, substance A functions as

• A) a coenzyme.
• B) an allosteric inhibitor.
• C) the substrate.
• D) an intermediate.
• E) a competitive inhibitor.

75) Choose the pair of terms that correctly completes this sentence: Catabolism is to anabolism as ________ is to ________.

• A) exergonic; spontaneous
• B) exergonic; endergonic
• C) free energy; entropy
• D) work; energy
• E) entropy; enthalpy
• 76) Most cells cannot harness heat to perform work because
• A) heat is not a form of energy.
• B) cells do not have much heat; they are relatively cool.
• C) temperature is usually uniform throughout a cell.
• D) heat can never be used to do work.
• E) heat must remain constant during work.
• 77) Which of the following metabolic processes can occur without a net influx of energy from some other process?
• A) ADP + i → ATP + H2O
• B) C6H12O6 + 6 O2 → 6 CO2 + 6 H2O
• C) 6CO2+6H2O→C6H12O6+6O2 D) amino acids → protein
• E) glucose + fructose → sucrose
• 78) If an enzyme in solution is saturated with substrate, the most effective way to obtain a faster yield of products is to
• A) add more of the enzyme.
• B) heat the solution to 90°C.
• C) add more substrate.
• D) add an allosteric inhibitor.
• E) add a noncompetitive inhibitor.
• 79) Some bacteria are metabolically active in hot springs because
• A) they are able to maintain a lower internal temperature.
• B) high temperatures make catalysis unnecessary.
• C) their enzymes have high optimal temperatures.
• D) their enzymes are completely insensitive to temperature.
• E) they use molecules other than proteins or RNAs as their main catalysts.

80) If an enzyme is added to a solution where its substrate and product are in equilibrium, what will occur?

• A) Additional product will be formed.
• B) Additional substrate will be formed.
• C) The reaction will change from endergonic to exergonic.
• D) The free energy of the system will change.
• E) Nothing; the reaction will stay at equilibrium.

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• Published: 24 August 2024

Polyphenol-stabilized coacervates for enzyme-triggered drug delivery

• Wonjun Yim 1 ,
• Zhicheng Jin 2 ,
• Yu-Ci Chang   ORCID: orcid.org/0000-0001-6997-9884 1 ,
• Carlos Brambila 2 ,
• Matthew N. Creyer 2 ,
• Chuxuan Ling 2 ,
• Tengyu He 1 ,
• Maurice Retout 2 ,
• William F. Penny 3 ,
• Jiajing Zhou   ORCID: orcid.org/0000-0001-5203-4737 2 &
• Jesse V. Jokerst   ORCID: orcid.org/0000-0003-2829-6408 1 , 2 , 4  

Nature Communications volume  15 , Article number:  7295 ( 2024 ) Cite this article

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• Drug delivery
• Molecular self-assembly

Stability issues in membrane-free coacervates have been addressed with coating strategies, but these approaches often compromise the permeability of the coacervate. Here we report a facile approach to maintain both stability and permeability using tannic acid and then demonstrate the value of this approach in enzyme-triggered drug release. First, we develop size-tunable coacervates via self-assembly of heparin glycosaminoglycan with tyrosine and arginine-based peptides. A thrombin-recognition site within the peptide building block results in heparin release upon thrombin proteolysis. Notably, polyphenols are integrated within the nano-coacervates to improve stability in biofluids. Phenolic crosslinking at the liquid-liquid interface enables nano-coacervates to maintain exceptional structural integrity across various environments. We discover a pivotal polyphenol threshold for preserving enzymatic activity alongside enhanced stability. The disassembly rate of the nano-coacervates increases as a function of thrombin activity, thus preventing a coagulation cascade. This polyphenol-based approach not only improves stability but also opens the way for applications in biomedicine, protease sensing, and bio-responsive drug delivery.

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Signal processing and generation of bioactive nitric oxide in a model prototissue

Introduction.

Coacervate droplets are cell-like compartments consisting of a condensed solution of macromolecules such as peptides 1 , copolymers 2 , RNAs 3 , and a diluted phase of the remaining liquid 4 . They formed instantly via liquid-liquid phase separation driven by non-covalent interactions such as electrostatic 5 , hydrophobic 6 , and hydrogen bonding 7 . The rich concentrations of biomolecules within the coacervates often mimic the physicochemical environments of living cells. Thus, coacervates have been extensively studied to understand the early stage of cell evolution 8 , 9 , 10 . The physical properties of coacervate droplets rely on the structural- and chemical- properties of their constituent building blocks 6 . However, the lack of membranes often leads to a rapid coalescence or collapse of the coacervate phases (i.e., poor stability) 11 , 12 . The absence of a physical membrane also limits their ability to mimic the selective permeability of cellular membranes 13 , 14 . These limitations challenge the promise of coacervate droplets for protocell models 14 , biomedicine 15 , drug delivery 16 , and biosensing 17 applications.

To address this stability issue, researchers have largely focused on developing hybrid protocell models consisting of coacervate-based interiors surrounded by membranes such as terpolymer 2 , phospholipids 18 , erythrocyte 19 , and polysaccharide 20 layers. These surrounding membranes either coat or encapsulate the coacervate droplet, which in turn enhances its stability. In the coating approach, a coacervate droplet is used as a template: Two opposite-charged building blocks initially form complex coacervates followed by the in-situ formation of membranes 18 , 19 , 20 , 21 . Alternatively, encapsulation of coacervates within liposomes is based on microfluidic techniques. Coacervates encased within liposomes have shown great potential in the development of a bio-responsive platform capable of reacting to changes in pH 22 , osmotic gradient 23 , and temperature 24 . However, these strategies often result in limited permeability of the surrounding membrane, which can hinder the penetration and/or release of large biomolecules 22 , 25 .

Our goal here is to establish a stable and enzyme-responsive coacervate platform. We first engineer a nano-sized coacervate made of bio-inspired peptides and the anticoagulant heparin. Heparin plays an important role in surgical and cardiovascular medicine due to its short half-life, reversible nature, and low cost 26 . However, heparin is difficult to manage and requires blood draws and central labs 27 ; therefore, the controlled release of heparin via the enzymatic activity of clotting factors is gaining interest 28 , 29 , 30 , 31 , 32 . Living organisms maintain hemostasis through precise molecular feedback regulations 33 . For example, vascular injury triggers a coagulation cascade process where clotting factors activate prothrombin to thrombin, transforming fibrinogen into insoluble fibrin by cleavage. Together with platelet activation, this process produces stable fibrin clots to prevent excessive bleeding 33 . We envision that by incorporating a feedback loop system within the coacervates, they could regulate heparin release based on thrombin activity. Increasing environmental thrombin levels would promptly trigger heparin release, while normal physiological thrombin levels would leave the coacervates intact—thus, there would be no risk of excessive bleeding 34 , 35 .

This work thus incorporates a thrombin cleavage site within the peptide used to make the nano-coacervate, resulting in the release of heparin as a function of concentration-dependent thrombin proteolysis. Importantly, we demonstrate enhanced coacervate stability via polyphenol-mediated supramolecular networks while maintaining their thrombin proteolytic activity. This structural and colloidal enhancement is obvious, clearly visualized by transmission electron microscopy (TEM)—they had exceptional stability in challenging conditions and various biofluids but could still specifically release the heparin cargo. The disassembly rate of nano-coacervates rapidly increased in response to thrombin proteolysis in human plasma. Overall, our approach of utilizing polyphenols to stabilize coacervates and preserve the bioactivity for enzymatic degradation is a simple yet powerful tool in the fields of biomedicine, biosensing, and enzyme-triggered drug delivery.

Nano-coacervates driven by a tyrosine and arginine peptide

The Mytilus edulis foot protein 5 (Mefp-5) in mussels contains repetitive DOPA and lysine (K) groups that provide positively charged residues with hydrophobic interactions 36 . This enables Mefp-5 to interact with a wide array of materials through either covalent or noncovalent interactions (Supplementary Fig.  1 ) 37 , 38 , 39 . The first step of designing our system was to determine whether a short peptide composed of tyrosine (Y) and arginine (R) could form a coacervate droplet with heparin (average M w : 15,000 Da) (Fig.  1a ). Heparin is a glycosaminoglycan with repeating sulfate units that provide negative charge and a polysaccharide structure for efficient binding with antithrombin 40 . Our previous studies revealed that heparin could assemble with small molecular dyes via strong electrostatic and hydrophobic interactions 29 , 41 suggesting that the repetitive YR sequence might also readily trigger the formation of coacervates. To test our hypothesis, we synthesized a short YRYR peptide (referred to as C2) and mixed C2 (0.05–1.5 mM) with heparin (50 U/ml). Upon interaction with heparin, the C2 peptide instantly formed coacervate droplets of varying sizes confirmed by dynamic light scattering (DLS) (Fig.  1b and Supplementary Fig.  2 ). Micro-sized coacervates exhibited a broad extinction spectrum, likely due to increased light scattering while the light absorption of nano-sized coacervates increased at more blue-shifted wavelengths (Fig.  1c and Supplementary Fig.  3 ).

a Schematic illustration of coacervate design using heparin and a YR-based short peptide. b DLS data of C2-heparin coacervates, showing their nano (198 ± 3.3 nm) or micro (>1 µm) sizes. c UV-vis spectra of nano- and micro-coacervates. Formation of coacervates as a function of different peptide ( d ) and heparin ( e ) concentrations. Six different peptide sequences (details described in Table  1 ) are examined to study the impact of the charge, concentration, number, thrombin recognition site, and length of YR-based peptides for heparin coacervation. The blue area indicates coacervate formation (i.e., phase separation). Red and blue dots indicate nano- and micro-sized coacervate formation, while empty dots represent no coacervate formation. f The photograph shows the increased turbidity as a function of coacervate formation. g High encapsulation efficiency of nano- and micro-coacervates. Eight dots indicate the encapsulation efficiency of eight independent coacervate samples. Stability test of nano-coacervates in different conditions ( h ) including PEG2000, citric acid, urea, Triton X-100, SDS, DMF, and DMSO, and different pH ( i ). j M-NTA images of nano- and micro- coacervates. Small blue dots represent monodispersed nano-coacervates, and large white dots indicate scattered micro-coacervates. The experiment was repeated three times independently with similar results. Data in ( h ) and ( i ) represent mean ± SD ( n   =  3). Figure 1/panel ( a ) Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

To further determine how many repeating YR units are needed to induce coacervation, YR, YRYR, and YRYRYRYR peptides were synthesized (referred to as C1, C2, and C3, respectively) as confirmed by matrix-assisted laser desorption/ionization (MALDI-TOF) (Supplementary Fig.  4 ). At a constant heparin concentration of 50 U/ml, C2, and C3 peptides formed coacervate droplets of different sizes from 70 nm to 1 µm while the C1 peptide was incapable of forming coacervates, indicating that heparin-based coacervation requires at least two YR units (Fig.  1d and Supplementary Figs  5 , 6 ). We further added six glycine (G) between YR sequences (YRG 6 YR, referred to as C4) to confirm the impact of charge density and peptide length. The results showed that both C2 and C4 require comparable peptide concentrations for coacervation, suggesting that the number of YR units (i.e., valence charge) plays a key role in coacervate formations rather than the steric bulk (extra glycine units) (Supplementary Fig.  7 ).

In addition, various heparin concentrations from 0.25 to 50 U/ml were combined with a constant peptide concentration of 1 mM, confirming that the coacervation relies on the number of YR units and heparin concentration (Fig.  1e and Supplementary Fig.  8 ). The formation of coacervate droplets led to an increase in turbidity, thus changing the color from transparent to white (Fig.  1f ). We also observed that the strong interactions between heparin and C2 peptide resulted in high loading efficiency of nano-coacervates (99.5–100%) (Fig.  1g and Supplementary Fig.  9 ). The nano-coacervates limit their growth and maintain their size and phase separation even under the centrifugation of 7 ×  g : There was no coalescence or merging.

To confirm the interactions governing the coacervate formation of the C2–heparin complex, nano-coacervates were incubated with PEG2000, citric acid, urea, Triton X-100, SDS, DMF, and DMSO, respectively. Triton X-100 and SDS can break non-ionic or ionic interactions; DMSO and DMF are organic solvents that can destroy pi-pi interaction 42 ; and urea can break the hydrogen bonding 43 . The nano-coacervates were disassembled in urea, Triton X-100, SDS, DMF, and DMSO conditions, indicating that electrostatic, pi-pi interaction, and hydrogen bonding were involved in the formation of C2–heparin coacervates (Fig.  1h and Supplementary Fig.  10 ). Nano-coacervates were stable at low pH (1–5), but they disassembled at high pH (over 9) due to the deprotonation of the guanidine group (Fig.  1i ). The isoelectric point of arginine is 10.8 44 . Finally, micro- and nano-coacervates were visually observed using multiple wavelength nanoparticle tracking analysis (M-NTA) 45 that further verified the narrow size distribution of nano-coacervates from DLS data (Fig.  1j , and Supplementary Fig.  6 , and Supplementary Movies  1 ‒ 3 ).

Enzyme-responsive coacervate droplets

Thrombin is a central enzyme in hemostasis and activates the fibrin network and platelets for blood clots when damaged tissue triggers factor VII (details described in Supplementary information) 46 , 47 . Heparin can prevent these clotting cascades because it contains saccharide units that bind to antithrombin, inactivating a number of coagulation enzymes 48 . We envisioned that if thrombin can cleave the peptide building block and disassemble the coacervates, then the coacervates could be an enzyme-responsive platform that can release the encapsulated heparin in response to thrombin proteolysis (Fig.  2a ). To achieve this, we added a thrombin cleavage site (LVPR ↓ GS) 49 in the middle of the C2 sequence: YRLVPRGSYR (referred to as C5) (Table  1 ). The thrombin proteolysis would result in fragment peptides that only contain one YR unit which is not sufficient for phase separation as depicted in Fig.  1d . Initially, we confirmed that the C5 peptide could form nano-coacervates with heparin, leading to an increase in turbidity (Fig.  1d and Supplementary Fig.  7 ).

a Schematic illustration of heparin release through disassembly of nano-coacervates driven by thrombin proteolysis. The released heparin binds antithrombin which induces thrombin inactivation. b Decreased turbidity of nano-coacervates as a function of thrombin concentrations. c Turbidity changes of C5- and C6-based nano-coacervates with and without thrombin (5 µM). d MALDI-MOF data before and after thrombin cleavage, confirming the mass peak of the parent (M w : 1307.91) and its fragment (M w : 845.63). The N-terminus of C5 was acetylated. e UV-vis and PL spectra of C7-encapsulated nano-coacervates before and after thrombin cleavage. The quenched PL signal of sulfo-Cy5.5 dyes activated as a function of nano-coacervates’ disassembly. f Time-dependent PL 670 nm changes driven by thrombin cleavage. g k cat /K M determination for C5 peptide cleavage driven by thrombin proteolysis. The thrombin (20 nM) was incubated with a fluorogenic substrate ([S] 0  = 0–30 µM, sequence shown on top in the panel box), and the product concentration at 30 min was used. Data was fit to the Michaelis–Menten equation (see Supplementary information  2 . 8 ). h Specificity test using different biological proteins including thrombin (Thr), bovine serum albumin (BSA), hemoglobin (Hemo), main protease of SARS-CoV-19 (M pro ), and α-amylase (50 U/ml). A sample without any proteins is referred to as a negative control. i Decreased absorbance of MB dye before and after the addition of thrombin. The disassembly of nano-coacervates released heparin, leading to a decrease in the absorbance of MB dye while intact nano-coacervates showed a negligible change in absorbance (Supplementary Fig.  17 ). j aPTT test of heparin and released heparin from the disassembly of nano-coacervates. Data in ( f ) and ( h ) represent the mean value of two independent samples. Data in ( b ), ( c ), ( g ), and ( j ) represent mean ± SD ( n   =  3).

The nano-coacervates were then incubated with various concentrations of thrombin from 0.05 to 2.5 µM. The turbidity of the nano-coacervates decreased due to thrombin proteolysis with higher concentrations of thrombin leading to rapid dissociation of the nano-coacervates (Fig.  2b and Supplementary Fig.  11 ). Notably, nano-coacervates composed of the scramble sequence (i.e., C6) showed negligible change in turbidity before and after thrombin incubation (Fig.  2c and Supplementary Fig.  12 ). We further confirmed the mass peak of the fragment peptide (845.63, YRLVPR) after thrombin cleavage via MALDI-TOF (Fig.  2d ).

In addition, the photoluminescence (PL) performance of C7-encapsulated nano-coacervates was examined upon thrombin proteolysis. We conjugated a sulfo-Cy5.5 dye with C5 peptide using an amine-NHS coupling (i.e., C7) and encapsulated C7 peptides within the nano-coacervates (details described in Supplementary Fig.  13 ). After C7 encapsulation, the PL signal of the C7 peptide was quenched, and the nano-coacervates exhibited a red-shifted absorption peak at 688 nm. This shift was likely due to increased intermolecular interactions, such as pi-pi stacking between tyrosines 50 , as well as electrostatic interactions between heparin and lysine within the nano-coacervates. Thrombin cleavage released sulfo-Cy5.5, recovering an absorption peak at 676 nm and its PL intensity at 700 nm (Fig.  2e ). The kinetics of PL activation increased as a function of thrombin concentration: Higher concentrations of thrombin led to a more rapid disassembly of nano-coacervates, promptly activating the PL signal (Fig.  2f ). Furthermore we determined the specificity constant (k cat /K M ) by thrombin using a fluorogenic substrate (Cy5.5–YRLVPRGSYRC–Cy3, referred to as C8) (Fig.  2g and Supplementary Figs  14 ‒ 15 ) and was 0.91 µM –1 s –1 , which is as fast as the thrombin-catalyzed conversion of human fibrinogen to fibrin (1.88 µM –1 s –1 ) 51 . The specificity of our system was further tested toward other proteins such as bovine serum albumin (BSA), hemoglobin (Hemo), SARS-CoV-2 main protease (M pro ), and α-amylase at the same enzyme concentration of 5 µM. No PL signal was activated in the presence of BSA, Hemo, and other enzymes (Fig.  2h and Supplementary Fig.  16 ).

Finally, a methylene blue (MB) assay was used to confirm the released heparin from the disassembly of nano-coacervates 52 . Heparin (from 0.125 to 5 U/ml) had a linear decrease in the absorption peak of the MB dye at 666 nm, subsequently causing a redshift of the peak to 566 nm due to the formation of heparin-MB complex (Supplementary Fig.  9 ). After incubation with thrombin, the disassembled samples were centrifuged to collect the supernatant. The supernatant obtained from the disassembled nano-coacervates decreased the absorption peak of the MB dye to 666 nm. Conversely, nano-coacervates without thrombin exhibited negligible changes in absorption, thus indicating that intact nano-coacervates did not release heparin (Fig.  2i  and Supplementary Fig. 17 ). In addition, the released heparin could prevent plasma coagulation as confirmed by an activated partial thromboplastin time (aPTT) test (Fig.  2j ). The C5 peptide only showed plasma coagulation due to lack of anticoagulant ability (Supplementary Fig.  18 ). Collectively, our coacervate-based heparin delivery offers an enzyme-responsive mechanism capable of releasing heparin in response to thrombin proteolysis for controlled anticoagulant therapy.

Polyphenol-stabilized nano-coacervates

A major drawback of using coacervate is their limited stability in biofluids. Human plasma, which contains diverse proteins, clotting factors, and ions, readily disrupts coacervate phases (Supplementary Fig.  19 ). We hypothesized that tannic acid (TA) could enhance colloidal and structural stability because multiple catechol groups in TA could create a strong supramolecular network with tyrosine 53 and polysaccharide 54 which are major structural components in the nano-coacervates. To verify this, the nano-coacervates were encapsulated with TA molecules of 0.05, 0.25, 0.5 mM under pH 8.5 (referred to as NC-TA 0.05, NC-TA 0.25 , and NC-TA 0.5 , respectively) (Fig.  3a ). These NC-TAs showed narrow size distributions (polydisperse index (PDI) ≤ 0.1), and similar hydrodynamic diameters (Fig.  3b ). The average diameter of each NC-TAs was 253.7 ± 9.4 nm (NC-TA 0 ), 221.8 ± 4.8 nm (NC-TA 0.05 ), 228.4 ± 7.1 nm (NC-TA 0.25 ), and 276 ± 4.3 nm (NC-TA 0.5 ), respectively. TA encapsulation resulted in a notable increase in the extinction value of NC-TAs in the near ultraviolet (UV) region, and the color of the sample changed from white to yellowish-brown (Fig.  3c and Supplementary Fig.  20 ). Fourier-transform infrared spectroscopy (FTIR) data evidenced the TA encapsulation as the appearance of C-O vibration (1320 cm –1 ) and 1, 3-disubstituted benzene rings around (1100–700 cm –1 ) (Fig.  3d ) 55 . To further understand TA encapsulation within the coacervates, NC-TAs were incubated at different pH and solvent conditions. NC-TAs remained stable until pH 10 but disassembled beyond 11 due to deprotonation (Fig.  3e ). In addition, DMF, DMSO, and SDS led to the disassembly of NC-TAs, suggesting that electrostatic and pi-pi interactions were involved in TA encapsulation (Fig.  3f ). NC-TAs showed higher stability in pH 9 and under urea compared to pristine nano-coacervates.

a Schematic illustration of TA encapsulation within the coacervates. DLS ( b ) UV-vis spectra ( c ) and FTIR ( d ) of NC-TAs. The brown region in ( c ) and ( d ) indicates the appeared peaks after TA encapsulation. Stability test of NC-TAs in different pH ( e ) and different conditions ( f ) including PEG2000, citric acid, urea, Triton X-100, SDS, DMF, and DMSO. g TEM image of NC-TA 0.13 . h Bright field (BF) and HAADF images of a single NC-TA 0.13 at different angles. Supplementary Figs  22 – 24 show multiple NC-TA 0.13 at different angles with low magnification. i , j EDX elemental mapping of a single NC-TA 0.13 , showing C, N, O, and S elements which are major components of heparin, peptide, and TA. The red-dotted line indicates the region used for the EDX mapping. The scale bar in ( h – j ) represents 100 nm. k SEM of micro-coacervates (i.e., MC-TAs). l Confocal image of MC-TAs encapsulating TA-coumarin conjugates. The yellow box indicates a single MC-TA with high magnification that highlights the evenly distributed fluorescent signal of TA-coumarin inside the MC-TA. This result reveals that TA is encapsulated within the coacervates. The scale bar represents 5 µm. Coumarin boronic acid was linked with hydroxyl groups in TA, forming a boronate ester, and the conjugates were purified using HPLC before encapsulation (Supplementary Fig.  28 ). Data in ( e ) and ( f ) represent mean ± SD ( n   =  3). The experiment in ( g – l ) was repeated three times independently with similar results.

We then attempted to image nano-coacervates before and after the TA encapsulation using TEM and scanning electron microscopy (SEM). NC-TAs maintained their size and spherical shape even in the vacuum condition confirmed by both TEM and SEM (Fig.  3g and Supplementary Fig.  21 ) while the nano-coacervates without TA collapsed and deformed during the drying process (Supplementary Fig.  22 ). Furthermore, we used tomography imaging at various angles ranging from –30° to 60° to illustrate the interface between the bottom of NC-TA 0.05 and the underlying substrate (i.e., TEM grid). Fig.  3h clearly shows the height of a single NC-TA 0.05 at 60°, indicating that TA molecules formed a rigid supramolecular network and enhanced the structural integrity of the nano-coacervates (Supplementary Fig.  23 ). High-angle annular dark field (HAADF) and energy-dispersive X-ray spectroscopy (EDX) were also utilized to confirm the elemental components of NC-TAs (Figs.  3i‒j ). EDX mapping revealed that C, N, O, and S signals were observed in a single NC-TA 0.05 which are components of TA, C5 peptide, and heparin (Fig.  3j and Supplementary Fig.  24 ).

We also discovered that polyphenol encapsulation can be applied to micro-sized coacervates (MC-TAs). The optical image illustrates the uniformly dispersed micro-coacervates after TA encapsulation (Supplementary Fig.  25 ). The spherical shapes and sizes of the dried MC-TA 0.05 were confirmed by the SEM technique (Fig.  3k and Supplementary Fig.  26 ). Notably, we observed significantly improved colloidal stability under the centrifugation of 3 ×  g . The coacervate droplets without TA showed a 98.7% decrease in turbidity while MC-TA 0.05 decreased by only 1.4% (Supplementary Fig.  27 ). Lastly, coumarin boronic acid was conjugated with TA for confocal imaging to verify TA encapsulation within coacervate droplets. HPLC was utilized to remove free coumarin dyes from TA-coumarin before encapsulation (Supplementary Fig.  28 ). Fig.  3l shows a uniformly distributed fluorescent signal of TA-coumarin conjugates from inside the MC-TAs. This result indicates that TA is encapsulated within the coacervates rather than being membrane-coated 10 , 56 .

Preserving enzymatic activity of nano-coacervates with enhanced stability

The formation of polyphenol networks within the coacervates significantly enhances stability; however, highly constructed supramolecular networks could adversely affect the proteolytic efficiency 6 (Fig.  4a ). To examine this, nano-coacervates encapsulated with various TA concentrations were incubated in NaCl for 1 h. We observed a less decrease in turbidity (T) as a function of increased TA encapsulation while nano-coacervates without TA were dissociated within the 30 s (Fig.  4b ): Turbidity (T after /T before ) of NC-TA 0 , NC-TA 0.17 , NC-TA 0.33 , and NC-TA 1 were 7%, 36%, 53%, and 92%, respectively. In contrast, high TA encapsulation led to a decrease in proteolytic activity. The turbidity changes of NC-TAs were measured after incubating different concentrations of alpha-thrombin (M w : 37.4 kDa) ranging from 0.06 to 1 µM. NC-TA 0.5 showed a reduced decrease in turbidity compared to NC-TA 0.25 when incubated with the same concentration of thrombin (Fig.  4c and Supplementary Fig.  29 ). NC-TA 1 exhibited negligible changes in turbidity, indicating that the excessive TA encapsulation could prevent thrombin-driven coacervate disassembly.

a Schematic illustration of a trade-off between stability and proteolysis-based disassembly of NC-TAs. NC-TAs increased stability ( b ) in NaCl as a function of TA encapsulations while reducing their proteolytic efficiencies ( c ). Thrombin was unable to dissociate NC-TA 1 . d Size profiles of NC-TA 0.13 in different biological environments. e Schematic illustration of monitoring either C7 peptide ( f ) or heparin-FITC ( g ) during disassembly of NC-TAs by thrombin. The left panels in ( f , g ) show a decrease in the PL activation rate of NC-TAs compared to nano-coacervates (i.e., NC-TA 0 ) due to improved stability in 50% human plasma. The right panels in ( f , g ) illustrate the addition of thrombin rapidly activates the PL intensity of C7 peptide or heparin-FITC, indicating proteolysis-driven heparin release. h Cell viability (blue) and ROS intensity (orange) of HUVEC incubating with PBS, TA, heparin, C5 peptide, and NC-TAs, respectively. i Prothrombin F1 + 2 peptide concentrations of NC-TA 0.13 TA, C5 peptide, and NC-TA 0.13 made of scramble peptide (i.e., C6) from whole human blood incubation. The inserted photo shows a strong blood clot from blood anticoagulation from NC-TA 0.13 (left) and scramble NC-TA 0.13 (right). j Residual thrombin activity in human serum and plasma. Human serum shows higher residual thrombin activity comparable to 42.5 nM of alpha-thrombin. The graphs on the right panel in ( j ) represent absorbance changes of nano-coacervates and NC-TA 0.13 before and after 1 h incubation in 50% human serum, showing higher stability of NC-TA 0.13 than pristine nano-coacervates. Data in ( c ), ( f ), ( g ), and ( i ) represent the mean value of two independent samples. Data in ( b ), ( d ), ( h ), and ( j ) represent mean ± SD ( n   =  3).

After identifying a critical TA encapsulation point for preserving thrombin proteolytic activity, we examined the colloidal stability of NC-TA 0.13 under various biological environments. The NC-TA 0.13 exhibited high colloidal stability in glutamine, glucose (5.6 mM), human albumin (0.6 mM), DPBS, NaOH (pH 10), 60 C°, NaCl (150 mM), fibrinogen (8.8 µM), 50% of Dulbecco’s Modified Eagle Medium (DMEM), serum, saliva, and urine (Fig.  4d and Supplementary Figs.  30 – 31 ). Both pristine nano-coacervates and NC-TAs containing either C7 peptides or heparin-FITC were incubated in 50% human plasma, respectively to examine improved stability. The quenched fluorescence of the C7 peptide and heparin-FITC was activated as a function of the disassembly of nano-coacervate (Fig.  4e ). NC-TA 0.13 exhibited a 3.3-fold decrease in the PL activation rate of C7 peptide than NC-TA 0 , indicating enhanced stability in human plasma. Simultaneously, thrombin could accelerate heparin release from NC-TA 0.13 (Fig.  4f ). The disassembly rate of NC-TA 0.13 increased by 4.2-fold upon the addition of thrombin (500 nM) which falls within the physiologic range of free thrombin concentration. Physiologic concentrations of free thrombin during coagulation reactions range over 500 nM 57 . We also monitored this disassembly process using heparin-FITC (Supplementary Fig.  32 ). NC-TAs exhibited a decrease in PL activation of heparin-FITC compared to pristine nano-coacervates in human plasma. Concurrently, the addition of thrombin rapidly increased the PL activation rate of heparin-FITC by 1.8-fold, recovering PL intensity within 10 min (Fig.  4g ). There was no fluorescence quenching of C7 peptides and heparin-FITC either by the background medium (i.e., human plasma) or by TA molecules (Supplementary Fig.  33 ). The release kinetics of heparin are different than the peptide because the concentration of coacervate samples and fluorescent dye-conjugates (C7 peptide and heparin-FITC) were different; the ratio could be tuned to control kinetics. We next examined the cell viability and cellular reactive oxygen species (ROS) levels of NC-TA 0.13 using human umbilical vein endothelial cells (HUVECs), respectively. NC-TAs and their structural components such as C5 peptide, heparin, and TA showed high cell viability (>83%) and minimal ROS intensities (Fig.  4h ). NC-TA 0.13 also exhibited low cytotoxicity against human embryonic kidney (HEK) 293 cells. NC-TAs led to minimal red fluorescence of propidium iodide (PI), which corresponds to cell viability of 95% (Supplementary Fig.  34 ).

We tested the anticoagulant performance of NC-TA 0.13 in whole human blood using a human prothrombin fragment 1 + 2 (F1 + 2) enzyme-linked immunosorbent assay (ELISA) kit. Fresh whole blood was collected using an EDTA-treated blood collection tube. Free heparin (0.6 U/ml), C6 peptide, TA, NC-TA 0.13, and scramble NC-TA 0.13 were incubated with whole human blood at the same concentration, respectively. Calcium chloride was then used to trigger blood clot formation. We observed a negligible difference in F1 + 2 formation between heparin and NC-TA 0.13 , confirming active blood anticoagulation driven by heparin released from NC-TA 0.13 (Fig.  4i and Supplementary Figs.  35 , 36 ). In contrast, a strong thrombus was observed in TA, C5 peptide only, and scramble NC-TA 0.13 . Note that scramble NC-TA 0.13 was comprised of a scramble C6 peptide, serving as a non-responsive control. Lastly, we examined the residual thrombin activity of human serum and plasma using a thrombin chromogenic substrate. Human serum exhibited at least 20-fold higher residual thrombin activity than plasma (Fig.  4j ). The residual thrombin activity in human serum was comparable to that of 42.5 nM of the alpha-thrombin (Supplementary Fig. 37 ). This difference arises because human plasma is obtained by anticoagulating blood, which prevents the clotting cascade, thus inhibiting thrombin formation. In contrast, human serum is collected by allowing blood to clot, during which thrombin is generated from prothrombin, resulting in higher residual thrombin activity. Nano-coacervates without TA showed a 56% decrease in absorbance at 500 nm due to residual thrombin activity while NC-TA 0.13 showed only an 18% decrease in 50% human serum, indicating enhanced stability through polyphenol encapsulations (Fig.  4j and Supplementary Fig.  37 ).

In summary, we report the self-assembly of YR-based peptides with heparin, forming coacervates through a combination of electrostatic, hydrogen bonding, and hydrophobic interactions. This assembly can produce a range of sizes from nano to micro-coacervates. In addition, the peptide building blocks involve a thrombin recognition site to incorporate a hemostasis feedback loop system within the coacervate for controlled heparin release. Increasing thrombin levels trigger the disassembly of the coacervates, rapidly releasing heparin, while the absence of thrombin leaves the coacervates intact.

The nano-coacervate was further stabilized via a polyphenol-mediated supramolecular network to improve its stability in human plasma. TA encapsulation improves the structural integrity of nano-coacervates as clearly visualized by TEM. Importantly, we demonstrated a critical TA concentration for preserving both thrombin proteolytic activity and colloidal stability. NC-TAs exhibited high stability under various biological conditions. Simultaneously, the disassembly rate of NC-TAs rapidly increased upon the addition of thrombin, leading to heparin release in human plasma. NC-TAs also feature bio-responsive anticoagulant performance in the whole human blood and high biocompatibility with HUVEC and HEK293 cells.

Coacervates containing catechol as structural building blocks have shown significant potential in drug delivery systems, particularly for gastrointestinal diseases 58 due to their strong adhesiveness 59 capable of prolonged retention in the gastrointestinal tract. Our nano-coacervates strengthened by polyphenols also showed superior coating ability on inert substrates and maintained high stability in whole human blood (Supplementary Fig.  38 ). Future work will incorporate coacervates on medical devices such as a drug-eluting stent for on-demand anticoagulant delivery. Studies on inflammatory aspects such as plasma viscosity, procalcitonin, and C-reactive protein levels‒as well as the elimination of particles from the circulation by phagocyte update or clearance in the kidney, spleen, and liver‒are needed to validate their value in translational nanomedicine. Taken together, our polyphenol-based platform to stabilize coacervates and preserve bioactivity may have a scope well beyond drug delivery, extending its applications to biomedicine, protease sensing, and hybrid protocell models.

A human blood specimen was collected from one male subject under approval from the institutional review board (IRB) of UC San Diego and the VA San Diego (#H170005). All subjects gave written informed consent. All work was done in accordance with the Declaration of Helsinki. We did not investigate sex as a biological variable because we are unaware of sex-based differences in thrombosis.

Experimental Details

Nano-coacervates preparation.

Briefly, 4 mg of the C5 peptides was dissolved in 3 ml of deionized water. Subsequently, 300 µL of heparin solution (100 U/ml) was mixed with 200- or 300- µL of the C5 peptides, immediately formulating nano- or micro-sized coacervates. The heparin concentration required to form coacervates depends on peptide concentration and the number of repetitive YR units in the peptide building block. The color of the solution became turbid once coacervation occurred. The size of coacervate droplets depends on the concentrations of the heparin and C5 peptides used for the coacervation. For example, 0.25 mM of the C5 peptide formed nano-coacervates at a constant heparin concentration of 50 U/ml while 0.5 mM of the C5 peptide formed micro-coacervates. The resulting product was purified by centrifugation at 3 ×  g for 10 min to remove any unreacted heparin or peptides. The pellet containing nano-coacervates was re-dispersed in MQ water for future use. However, the micro-sized coacervates were deformed, and the coacervate phase disappeared after centrifugation at 3 ×  g . All peptide sequences were synthesized using an AAPPTEC peptide synthesizer (see  Supplementary information ).

Polyphenol Encapsulation within Coacervates

The nano-coacervates were re-dispersed in 200 µL of bicine buffer at pH 8.5. Subsequently, the desired amount of TA in MQ water was added to the nano-coacervates, and the mixture was gently shaken (400 rpm) at 37 °C for 6 h. During this process, the color of the solvent was changed from white to yellowish due to TA oxidization. The resulting product was once again centrifuged at 3 ×  g for 10 min to remove any unreacted TA molecules. The 0.03, 0.17, 0.33, and 1 mM of TA were incubated with nano-coacervates containing 1 mM of C5 peptide. Note that excess amounts of TA could lead to the formation of solid precipitates.

The micro-coacervates were first prepared by mixing 300 µL of heparin (100 U/ml) with 300 µL peptide (1.3 mg/ml) followed by the addition of TA molecules with bicine buffer overnight. The resulting product was centrifuged at 3 ×  g for 10 min to remove any unreacted TA molecules. The pellet was re-dispersed in MQ water for future use. Note that excessive TA molecules can trigger solid aggregates (Supplementary Fig.  25 ).

Sulfo-Cy5.5–Labeled C5 Peptides (i.e., C7)

Sulfo-Cy5.5-NHS was coupled with the free amine from the N-terminus of the C5 peptide to encapsulate sulfo-Cy5.5 dye within the nano-coacervates (see Supplementary information  2.2 ). Briefly, the desired amount of the C5 peptide was dissolved in DMSO with 1% v/v triethylamine wrapped with aluminum foil. Subsequently, sulfo-Cy5.5-NHS was added to the C5 peptides at a 1:1 molar ratio under generous stirring for 3 h. After the amine-NHS couplings, the final product was fully dried using a vacufuge. The resulting product was re-dispersed in 50% ACN for HPLC purification. MALDI-MOF MS was used to confirm the molecular weight of the final product (Supplementary Fig.  13 ).

For the encapsulation of C7, the nano-coacervates were dispersed in bicine buffer at pH 8.5, followed by the addition of C7 peptides under generous for 3 h. The resulting product was centrifuged at 3 ×  g for 10 min to remove any unencapsulated C7 peptides. The pellet was re-dispersed in MQ water for future use.

Thrombin Proteolysis of Nano-Coacervates

Briefly, 1 mM of the nano-coacervates was incubated with various concentrations of α-thrombin ranging from 0.03 to 4 µM in 20 mM Tris-HCl buffer solution (pH 7.4, NaCl 150 mM). The mixture was immediately transferred into a 96-well plate, and the light absorption at 500 nm was measured at 37 °C for 1 h. The fragment solution was desalted by using a C18 column (5 µm, 9.4  \(\times\)  250 mm) and then applied MALDI-TOF to confirm the cleavage site. Mass peaks shown in Fig.  2d are from the acetylated C5 parent and its fragment peptides.

Likewise, the C7-encapsulated nano-coacervates and NC-TA 0.13 were incubated in 50% human plasma containing the alpha thrombin (final concentration = 500 nM) at 37 °C. The mixture was immediately transferred into a 96-well plate, and the fluorescence signal at 670 nm was measured at 37 °C for 3 h.

Stability test of NC-TAs

Briefly, the nano-coacervates encapsulated with TA molecules (0.03–1 mM) were incubated with 1 M NaCl for 1 h at room temperature in a 96-well plate. The absorbance from 300 to 900 nm was measured with a step size of 2 nm before and after the incubation. The absorbances at 500 nm before and after incubation were used to calculate turbidity changes (Turbidityafter/Turbiditybefore).

For the size measurements, NC-TA 0.13 was incubated for 1 h under various conditions including fibrinogen, glucose, glutamine, acetone, methanol (MeOH), citric acid (pH 2), DPBS, NaOH (pH 10), 60 °C, human albumin, NaCl of 150 mM, 50% of human serum, human urine, human saliva, and Dulbecco’s modified Eagle’s medium (DMEM), respectively. After incubation, the resulting samples were centrifuged at 3 ×  g to replace the medium with MQ water for DLS measurement. The average size was calibrated using three independent replicates. The human saliva, serum, and urine samples were purchased from Innovative Research.

C7 peptide/Heparin-FITC encapsulation and fluorescent monitoring in biofluids

Briefly, NC-TAs were gently mixed with either C7 peptide (20 µM) or heparin-FITC (150 µM) for the encapsulation. The desired dye-conjugates were encapsulated within NC-TA 0 (i.e., nano-coacervates), NC-TA 0.07 , and NC-TA 0.13 , respectively with the same number of coacervates under generous stirring overnight. Note that the coacervate concentrations used for Figs.  3f and g are different. Following encapsulation, each sample was purified using centrifugation at 3 ×  g for 10 min. Subsequently, the samples were transferred to a 96-well plate at 37 °C for PL measurement.

For fluorescence monitoring, NC-TAs encapsulating with either C7 peptide (Fig.  3f ) or heparin-FITC (Fig.  3g ) were incubated in 50% of human plasma in a final volume of 100 µL. The mixture was directly transferred to a 96-well plate at 37 °C, and the PL intensity at 670 nm (for C7 peptide) and 520 nm (for heparin-FITC) was recorded for 1 h with 1 min intervals. The activated PL signal indicates the disassembly of nano-coacervates in 50% human plasma. Notably, there was no self-PL quenching of C7 peptide or heparin-FITC in biofluids (Supplementary Fig.  33 ). The slope is referred to as the PL activation rate. Normal pooled plasma and normal pooled serum were purchased from Innovated Research.

Cytotoxicity test and ROS detection of HUVEC

HUVEC cells (ATCC, PCS-100-100) were cultured in a vascular cell basal medium with an endothelial cell growth kit. Cell cultures were incubated under 5% CO 2 at 37 °C. Cells were passaged when they reached 75–80% confluency using 0.25% trypsin for primary cells. DPBS and cell lysis buffer were used for negative and positive controls of healthy and dead cells.

For the cytotoxicity experiments, HUVEC cells were seeded overnight in a 96-well plate at a concentration of 10,000 cells/well. Subsequently, PBS, lysis buffer, C5 peptide, TA, nano-coacervates, NC-TA 0.25 , NC-TA 0.5 , and NC-TA 1 were co-incubated with HUVEC at an equal concentration of 0.16 mM for 12 h, respectively. A resazurin assay was used to analyze the cytotoxicity of nano-coacervates, NC-TA 0.25 , NC-TA 0.5 , and NC-TA 1 following a protocol. After 4 h incubation with resazurin, cell viability was calibrated by measuring the subtracted background absorbance of each well at 600 nm from resazurin absorbance at 570 nm. The absorbances of experimental wells were compared to those of the controlled wells containing healthy and dead cells.

HUVEC cells were seeded overnight in a 96-well plate for reactive oxygen species (ROS) detection test using a DCF-DA kit. Subsequently, PBS, lysis buffer, C5 peptide, TA, nano-coacervates, NC-TA 0.25 , NC-TA 0.5 , and NC-TA 1 were co-incubated with HUVEC at an equal concentration of 0.16 mM for 3 h, respectively. N-acetyl Cysteine and pyocyanin were added as the negative and positive controls. The fluorescence of experimental wells was compared to those of the controlled wells containing negative and positive controls. All experiments were performed in triplicate to measure the average and standard deviations.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The data generated in this study are provided in the  Supplementary Information / Source Data file. The data supporting the findings of this study are also available from the corresponding author upon request.  Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Institute of Health (DP2 HL137187 and R21 GM153048). W.Y. acknowledges the Schmidt Science Fellowship and  the Diversity Fellowship from the UCSD Materials Science and Engineering program. W.Y. and C.B. thank the GEAR mentorship program at UC San Diego. This work was partly performed at the San Diego Nanotechnology Infrastructure (SDNI) of UCSD, supported by the National Science Foundation (Grant ECCS-2025752, and #2242375). The author acknowledges the use of Biorender software for scientific schematics (Fig.  1a ).

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W.Y. and J.V.J. conceived the idea and developed the materials. W.Y. designed and performed major peptide synthesis and experimental works. Z.J. performed confocal imaging and ELISA. Z.J., Y.-C.C., M.N.C., T.H., Y.L., W.F.P., and J.Z. helped with other experimental work, sample collection, and scientific discussions. Y.-C.C. C.B. helped with peptide synthesis and purification. C.L., T.H., and M.R. helped with material characterizations. W.Y. and J.V.J. drafted the manuscript with input from all authors.

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Yim, W., Jin, Z., Chang, YC. et al. Polyphenol-stabilized coacervates for enzyme-triggered drug delivery. Nat Commun 15 , 7295 (2024). https://doi.org/10.1038/s41467-024-51218-8

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DOI : https://doi.org/10.1038/s41467-024-51218-8

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