5.16, Core practical 12 - Temperature, enzyme rate and Q10

Temperature and enzyme molecules

Temperature affects both the speed of molecular encounters and the structure needed for catalysis.

An enzyme's active site binds its substrate and enables a reaction with a lower activation energy. At a low temperature, enzyme and substrate molecules have less kinetic energy. Encounters are less frequent, and fewer encounters have sufficient energy for reaction. Raising temperature within a suitable range increases successful encounters and the number of reactions per second. The enzyme does not supply the thermal energy or become used up by each reaction.

At sufficiently high temperatures, interactions maintaining the enzyme's folded structure can be disrupted. The proportion of active enzyme may fall, so the measured rate can decrease despite faster molecular motion. Thermal damage depends on both temperature and exposure time. There is no single temperature at which every enzyme denatures, and denaturation is not the normal breaking of all its peptide bonds. A low rate after cooling a strongly heated enzyme can support lasting damage, whereas a chilled enzyme often becomes more active when rewarmed.

The observed optimum temperature is the temperature giving the greatest measured activity under the stated assay conditions. It depends on the enzyme, pH, substrate conditions and measurement duration. It is not a universal 37 °C or a boundary below which no structural changes can occur. Thermostable enzymes can retain activity at temperatures that inactivate other enzymes. A falling activity curve is consistent with heat inactivation, but an experiment must also exclude temperature-dependent measurement artefacts before attributing the whole change to denaturation.

Effects on living organisms

A change in enzyme activity can alter an organism's energy supply, growth and survival, although a whole organism is more complex than one enzyme in a tube.

In plants, enzyme-controlled photosynthesis, respiration and biosynthesis respond to temperature. Warming a cold seedling can increase metabolic rates and growth when water, light and nutrients are adequate. Warming does not guarantee greater biomass: respiration can increase the use of respiratory substrates, and photosynthesis can become limited by light, carbon dioxide or water. Heat damage to proteins and membranes can reduce photosynthesis and growth. A plant's response therefore depends on the balance between carbon gain, carbon use and damage.

In animals, respiration supplies ATP for processes including movement and growth. In an ectotherm, body temperature is strongly influenced by the environment; warming within a tolerated range can accelerate enzyme-controlled metabolism and increase oxygen and food requirements. If oxygen delivery or food supply cannot meet demand, performance need not improve. Endotherms regulate body temperature, so an increase in air temperature need not cause an equal increase in tissue temperature. Maintaining a suitable internal temperature protects metabolic function; extreme overheating can disrupt proteins and organs.

In microorganisms, enzymes control nutrient breakdown, respiration and synthesis of new cell material. Within a suitable range, warming can accelerate these reactions and cell division when nutrients and other conditions permit. Chilling food often slows microbial metabolism and growth; it does not necessarily kill microorganisms. A sufficiently severe heat treatment can inactivate enzymes and damage other cell components. Different microorganisms are adapted to different thermal conditions, so one culture's rate curve cannot stand for all microbes.

These mechanisms help explain temperature effects on development and life cycles studied in Core practical 13. A rapid response during one lifetime is not, by itself, evidence of evolutionary change in a population.

Preparing a measurable reaction

Core practical 12 needs a signal that follows the reaction over time while temperature is deliberately varied.

Use catalase from a fresh plant extract to catalyse hydrogen peroxide breakdown:

A freely moving gas syringe connected to the reaction vessel records oxygen volume. At a common gas temperature and pressure, this volume is proportional to the amount of gas. It is an oxygen-production measure, not a direct measurement of enzyme concentration. A pressure sensor is an alternative quantitative instrument, but its signal also depends on gas temperature and headspace; pressure cannot simply be relabelled as volume.

For a supervised starting method, homogenise 100 g of soaked peas with pH 7 buffer, strain through muslin and make the filtrate up to 300 cm³ with the same buffer. The teacher operates any cutting or blending apparatus. Keep one mixed fresh batch cool until portions are needed and gently remix before withdrawing them. Its catalase activity is not a known molar concentration; record the preparation and call it a defined extract batch.

Pilot a suitable extract dilution and dilute peroxide at the fastest expected condition. For example, combine 10.0 cm³ extract dilution with 10.0 cm³ of 0.200 mol dm⁻³ peroxide, giving a final peroxide concentration of 0.100 mol dm⁻³ in 20.0 cm³. Use a syringe of suitable capacity, such as 50 cm³, and dilute the enzyme consistently if the earliest change is too fast to resolve. The pilot sets a common enzyme dilution for the whole temperature series, rather than changing concentration at each temperature.

Glassware and measured-volume pipettes allow controlled dilution. A twofold serial dilution transfers 20.0 cm³ stock into 20.0 cm³ buffer, mixes thoroughly, then transfers 20.0 cm³ of that dilution into another 20.0 cm³ buffer. These are half-stock and quarter-stock. Mix before each transfer, use clean apparatus and label each vessel. An early dilution error is inherited by subsequent transfers. Prepare enough of the selected dilution for all fresh repeats; record the final reaction dilution as well as the bottle concentration.

Qualitative reagents and an alternative enzyme system

Collecting oxygen does not itself identify a biological molecule with a qualitative reagent. A separate amylase–starch comparison supplies that technique honestly. Teacher-prepared fresh 0.5% starch means 0.50 g made up to 100 cm³, with heating needed to disperse soluble starch followed by cooling before use. The teacher prepares a fresh 0.5% amylase solution to avoid airborne enzyme powder, and supplies dilute iodine in potassium iodide solution; potassium iodide helps iodine dissolve. For example, making 10.0 cm³ of 0.100 mol dm⁻³ iodine stock up to 100.0 cm³ gives a 0.0100 mol dm⁻³ test reagent. Wear eye protection, avoid skin contact and rinse splashes as directed: iodine stains and can irritate eyes.

On a white spotting tile, a separate starch-plus-iodine control should become blue-black; water plus iodine should retain the iodine's orange-brown colour. Equilibrate 2.0 cm³ starch, 2.0 cm³ amylase and 1.0 cm³ suitable pH 6 buffer separately, then mix and start timing. Transfer a small consistent sample to a fresh iodine drop every ten seconds, using clean equipment and keeping iodine out of the reaction tube. The first orange-brown test indicates starch below the test's detection threshold, not the identity of a sugar product. Record the previous positive time as well as the first negative time.

This endpoint method can compare a fixed amount of substrate reaching the same threshold. Its reciprocal time is an interval-speed proxy; it is not a measured initial tangent. Continuous calibrated concentration measurements would be needed to infer an initial amylase rate. The main catalase method below supplies the required product–time evidence.

Controlling the temperature investigation

A valid temperature investigation changes the temperature of the reacting mixture while keeping other causes of rate differences controlled.

Predict an increase in initial rate over a suitable low-temperature range, followed by lower activity after sufficiently strong heating. Use a pilot to choose a feasible range, for example 20, 30, 40, 50 and 60 °C. Ten-degree spacing provides direct Q10 comparisons; add closer temperatures around an apparent peak if its location matters. The hottest tested condition is not automatically the optimum.

1. Set up thermostatically controlled water baths. Check with a thermometer or calibrated temperature probe, rather than trusting the dial. Record instrument resolution and stated uncertainty. Use the same measured enzyme and peroxide volumes, buffer and extract batch throughout.
2. Place the enzyme portion and peroxide portion separately in the bath for a fixed five-minute equilibration, or a pilot-verified time sufficient for both to reach the bath temperature. Keep exposure time the same: holding enzyme at high temperature for twenty minutes in one trial and five in another changes possible heat inactivation. Check actual reagent temperature with clean probes or matched spare portions.
3. Use a reaction flask with a small internal vial so both portions can equilibrate without contacting each other. Fit the bung, connect the gas syringe and let the apparatus reach a stable baseline before mixing. Keep the collecting syringe at the same monitored room temperature for all trials. A suitable length of delivery tubing at that collection temperature allows gas to approach the common collection temperature; validate that it does not create an unacceptable response delay. Never seal an apparatus and then warm its headspace while counting expansion as product.
4. Record the initial syringe reading. Tilt the vessel to combine the solutions, mark first contact as time zero and mix consistently. Keep the reacting vessel in its bath. Check actual mixture temperature at the start and end using a sealed probe port if available, or matched parallel mixtures; do not open the gas vessel during a run. Record drift and any mixing/sensor delay.
5. Follow oxygen volume at closely spaced early times and continue to show whether the curve bends. A pilot might support readings at 0, 5, 10, 20, 40, 60 and 100 s; a faster reaction needs closer early intervals. An electronic gas-volume sensor/data logger can record more frequently. Check calibration, sampling interval and sensor response before accepting its output. Save the complete original time series and the recorded temperature trace.
6. Disconnect safely before emptying, clean the apparatus and start each repeat with fresh measured enzyme and substrate. Obtain at least three independent reactions at each temperature. Several readings during one reaction are a time course, not independent repeats. Interleave temperatures or run a reference temperature periodically to reveal extract ageing; keep equilibration duration equal for every portion.
7. Run a no-enzyme blank at each temperature, replacing the enzyme aliquot with equal buffer volume. This checks peroxide breakdown and physical gas-volume changes without added enzyme. If the blank has a substantial time-dependent signal, investigate rather than assuming all gas is enzyme activity. Apply a blank correction only when its behaviour justifies it, preserving both original traces and the correction.

Wear eye protection and laboratory clothing. Peroxide can harm eyes and skin; the teacher prepares the dilute stock, checks its current supplier information and the centre's risk assessment, and deals with splashes using the laboratory procedure. Enzyme-containing material should not be inhaled or tasted. Handle hot baths with appropriate tongs or protection, use stable racks, and keep electrical equipment dry. Oxygen supports combustion: keep flames away. A blocked delivery tube or restrained syringe can create pressure, so check the outlet and plunger before use and never force the bung. Plant extract avoids use of live animals, but minimise waste and dispose of peroxide and plant material as directed.