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10: Enzymes and pH Buffer

Last updated

Apr 19, 2025
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- 9: Dilutions and Spectrophotometry
- 11: Creating Buffer Solutions

Victor Pham
Irvine Valley College

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Learning Objectives

Students should understand the fundamental concepts of enzyme function, pH, and buffer systems. The goal is to prepare students for hands-on experimentation by reinforcing theoretical knowledge and ensuring they can make informed observations.

Pre-Lab Questions

What are enzymes, and how do they facilitate biochemical reactions?
Explain the role of activation energy in enzymatic reactions.
What is equilibrium in the context of enzyme activity, and how do enzymes affect reaction rates?
Define pH and explain why pH stability is important for enzyme function.
What are buffers, and how do they help maintain pH stability in biological systems?
How does the Henderson-Hasselbalch equation help determine buffer capacity and pH adjustments?

Note

Review the concept of enzyme-substrate interactions.
Read about the effects of pH on enzyme activity and how buffers stabilize pH.
Familiarize yourself with the procedure for calibrating and using a pH meter.
Review the Henderson-Hasselbalch equation and perform sample calculations for buffer preparation.
Predict how changes in pH might affect enzyme activity and buffer efficiency.

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Chapter Ten
Enzymes and pH Buffer

In this Chapter, we're embarking on enzymes, those remarkable molecular machines that drive the biochemical processes essential for life. But before we delve into the details of how enzymes work, let's first understand the fundamental concept of substrate conversion into products. Imagine you're in a classroom, attempting to throw paper balls into a trash can. Now, think about the chances of success if you randomly toss the paper balls without aiming properly. Not very high, right? Well, in biochemical reactions, substrates must align correctly for reactions to occur effectively. Enzymes come to the rescue by guiding substrates into the right positions, much like someone helping you aim those paper balls directly into the trash can, increasing the likelihood of successful conversions.

However, it's crucial to understand that enzymes don't change the final outcome of reactions. Instead, they accelerate the reaction to equilibrium, which represents the peak point of substrate and product conversion. To illustrate this concept, envision tossing a bunch of papers into a trash can. However, if the trash is already full, no matter how many papers you attempt to toss in, they will simply fall off. This scenario mirrors the concept of equilibrium in enzymatic reactions, where substrate and product conversion reaches its peak. Furthermore, the reduction of transition energy can be likened to cycling from point A to point B. Imagine you're riding a bike from point A to point B. Without an enzyme, it's akin to pedaling uphill – a strenuous task. However, with the assistance of an enzyme, it's like cycling on a flat road – significantly easier. Enzymes achieve this by reducing the activation energy needed for reactions to proceed, facilitating the transition to equilibrium. Nonetheless, it's important to note that enzymes do not alter the equilibrium state itself.

Now, let's transition to our next topic: pH and its critical role in enzyme function. Enzymes, like many other molecules and proteins, are sensitive to changes in pH. Most life forms thrive within a relatively narrow pH range, and extreme pH levels can lead to protein denaturation and precipitation. This highlights the importance of maintaining pH balance within living systems. But how do living organisms maintain pH balance, especially in the face of dramatic pH changes? Buffers – nature's pH regulators. Buffers are solutions capable of resisting changes in pH by absorbing or releasing hydrogen ions (H+) or hydroxide ions (OH-).

To understand buffers better, let's compare strong acids like hydrochloric acid (HCl) to weak acids like acetic acid. When you add HCl to water, it fully dissociates, rapidly lowering the pH. In contrast, weak acids like acetic acid only partially dissociate, allowing them to maintain pH stability by absorbing excess H+ ions. This buffering capacity is essential for preventing drastic changes in pH, akin to a trashcan absorbing additional trash to keep the environment clean.

However, it's crucial to recognize that even buffers have their limits. Every weak acid or base has a specific buffer zone, akin to holding a trash can steady. Within this buffer zone, the molecule can effectively maintain pH stability, much like keeping the trash can balanced without spilling its contents. However, when you go beyond this buffer zone, it's like piling too much trash onto the can. Eventually, the can tilts, and all the trash spills out. Similarly, when adjusting the pH from 7 to 6, adding slightly more acid can cause the pH to plummet to 2 – a clear indication of surpassing the buffer zone's capacity to stabilize pH.

Now, let's apply our understanding of pH and buffers to the laboratory setting. Suppose we want to manipulate pH to create optimal conditions for enzymatic reactions. In this scenario, we can use the Henderson-Hasselbalch equation to calculate the precise amount of acid needed to achieve a target pH. For example, let's say we mixed 1.67mL of 1M acetate with 1mL of 1M acetic acid, with a pKa value of 4.76. By plugging these values into the Henderson-Hasselbalch equation, we can determine the resulting pH of the solution.

Step 1: Calculate the moles of acetate (buffer):

Buffer = Volume x Concentration

= 0.00167 L x 1 mol/L

= 0.00167 mol

Step 2: Calculate the moles of acetic acid (weak acid):

Acid = Volume x Concentration

= 0.001 L x 1 mol/L

= 0.001 mol

Step 3: Substitute the values into the Henderson-Hasselbalch equation:

pH = pKa + log([A-]/[HA])

pH = 4.76 + log(0.00167/0.001)

Step 4: Perform the logarithmic calculation:

pH = 4.76 + log(1.67)

pH ≈ 4.76 + 0.2228

pH ≈ 4.983

But what if we want to adjust the pH to a specific value, such as pH 5? How much volume of 1M acetic acid should you add to mix with 1.67mL of 1M acetate to obtain pH 5? By rearranging the Henderson-Hasselbalch equation and solving for the acid concentration, we can calculate the volume of acid needed to achieve the desired pH. In this case, we find that only 1.05mL of acid is required, demonstrating the precision and utility of buffer calculations in laboratory experiments.

Step 1: Substitute the values into the Henderson-Hasselbalch equation:

pH = pKa + log([A-]/[HA])

5 = 4.76 + log[(0.00167L)(1mol/L)/(1mol/L)(A-L)]

Step 2: Rearrange the equation to solve for the concentration of acetic acid (HA):

5 - 4.76 = log(0.00167/A-L)

0.24 = log(0.00167/A-L)

Step 3: Rewrite the equation in exponential form:

100.24 = 0.00167/A-L

Step 4: Solve for the concentration of acetic acid (HA):

A-L = 0.00167/100.24

≈ 0.00167/1.738

A-L ≈ 0.00096

Step 5: Divide 0.00096M by the Molarity of acid to determine the volume of acetic acid needed:

A-L ≈ 0.00096 ≈ 0.00096 L or 0.96mL

In conclusion, enzymes are the unsung heroes of biochemical reactions, driving essential processes with finesse and precision. Yet, their activity is intricately linked to factors such as pH, which can profoundly influence enzymatic function. Buffers play a crucial role in maintaining pH stability, ensuring optimal conditions for enzyme activity. By understanding the interplay between enzymes, pH, and buffers, we unlock the secrets of life's biochemical complexity and pave the way for groundbreaking discoveries in science and medicine.

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LAB ACTIVITY - PART 1 - pH meter Calibration and Verification

Why it's crucial to calibrate your pH meter and verify it with known standards? pH meters measure the acidity or alkalinity of a solution based on the voltage generated by a pH-sensitive electrode. Over time, factors such as electrode wear, contamination, or drift can affect the accuracy of readings. Calibration ensures that the pH meter provides accurate and reliable measurements. Verifying the pH meter with known buffer standards validates its accuracy and reliability. Known buffer solutions with precise pH values serve as reference points for verifying the performance of the pH meter. Verification with known standards is essential for quality control purposes, especially in laboratory settings. It ensures that the pH meter meets quality assurance standards and adheres to regulatory requirements for accurate pH measurements.

Instruction: Preparation Before Using the pH meter:

Turn on the pH meter and allow it to warm up.
Rinse the pH electrode with distilled water to remove any residue or contaminants.
Immerse the electrode in a storage solution or distilled water to hydrate the sensitive electrode until you are ready to use it.

Instruction: Using the Probe:

Gently wipe off excess liquid, but only dap to dry the sensitive electrode.
Carefully submerge the electrode into the sample solution.
Allow the pH reading to stabilize, and write down the pH value.
1. If it’s not stabilizing within 1 minute, go ahead and write down what you see.

Instruction: Cleaning After Use:

After measurements are complete, remove the pH electrode from the sample solution.
Rinse the electrode with distilled water to remove any residual sample solution.
Gently wipe off excess liquid, but only dap to dry the sensitive electrode.
Store the pH electrode in a storage solution or distilled water until the next use.

Instructions: Calibrate Your pH Meter:

Prepare buffer solutions with pH values of 4.01, 7.00, and 10.01.
Immerse the pH electrode of the meter into each buffer solution sequentially.
Follow the manufacturer's instructions to calibrate the pH meter using the provided calibration controls.
Ensure that the meter is properly calibrated and stable before proceeding to the next step.

Instruction: Verify with Buffer Solutions (AFTER CALIBRATION IS COMPLETED)

Re-measure the same buffer solution (4.01, 7.00, and 10.01), without calibration
Immerse the pH electrode into the buffer solution with a pH of 4.01.
Record the pH reading displayed on the meter.
Repeat the process with buffer solutions of pH 7.00 and 10.01, recording the pH readings each time.
Determine Absolute Error:
1. Absolute Error = |Measured pH - Known pH|
2. Tape the absolute error data on the pH meter.

Summary instruction:

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LAB ACTIVITY - PART 2 - pH Calculation and measurement

Materials:

1M Acetic acid
1M Sodium Acetate

Theoretical Calculation:

Calculate and determine the quantities of 0.1M acetic acid needed to achieve the theoretical pH values of 4.76, 6, 7, and 8 given a volume of 10mL 0.1M Sodium acetate.
- Utilize the Henderson-Hasselbalch equation.

Actual Experiment:

Dilute the 1M Acetic acid and 1M Sodium Acetate
Mix in the calculated amounts of 0.1M acetic acid with 10 mL of 0.1M Sodium acetate in a small beaker for each target pH value (4.76, 5, 5.5, and 6)
Use a magnetic stir to mix to achieve a homogeneous solution.
Use a calibrated pH meter to measure the pH of the mixed solution for each solution.
Record the measured pH values obtained.

Theoretical pH	0.1M Sodium acetate (mL)	0.1M acetic acid (mL)	Actual pH
4.76	10
6	10
7	10
8	10

---------------------------------------------

LAB ACTIVITY - PART 3 - Skill Evaluation 3 (20 points)

Materials:

1M Acetic acid
1M Sodium Acetate

Calculation:

Calculate and record the exact volumes of 0.1M acetic acid required to achieve pH values of 5.8 given a volume of 20mL of 0.1M Sodium acetate.

Testing:

Dilute 1M Sodium acetate and 1M acetic acid solutions to 0.1M concentrations.
Measure out and mix in the exact volumes of the 0.1M Sodium acetate and 0.1M acetic acid solutions determined from the calculations to obtain a pH value of 5.8.

Grading:

The instructor will measure and record your pH.
Deduct the percent error from the total score as a grading criterion.
- If the percent error is 5%, then the total score will be 15/20.

-------------------------------------------

LAB ACTIVITY - PART 4 - Practicum 1 (60 points)

Dilution Activity (20 points):

Materials:
- Bottle of 0.5M stock CuIISO4 solution
- Unknown CuIISO4 solution was created by the instructor (100mM to 250mM).
- Cuvette and spectrophotometer
Goal: Determine the concentration of the unknown solution
- Create a 2-Fold serial dilution
- Measure your dilution and the unknown via Spectrophotometry.
- Create a standard curve graph. Calculate the concentration of your unknown.
Evaluation: The percent error from your calculated concentration and the actual concentration will be deducted from the total score.
- If the percent error is 5%, then the total score will be 15/20.

--------------------------

Dilution Re-creation (20 points):

Materials:
- Bottle of 0.5M stock CuIISO4 solution
- Distilled water.
- Cuvette and spectrophotometer
Goal: Recreate the unknown solution.
- Apply C1V1=C2V2 to calculate the volume of 0.5M CuIISO4 and water to mix to obtain the predicted unknown concentration from Part 1.
- Dilute the 0.5M CuIISO4 with water to have the same concentration as Part 1.
  - Make sure the total volume is 1 mL in a cuvette.
- Submit the cuvette to your instructor.
Evaluation: The percent error from your recreated absorbance and your previous absorbance will be deducted from the total score.
- If the percent error is 5%, then the total score will be 15/20.

Summary instruction:

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pH Activity (20 points): pH meter is not allowed during this examination

Materials:
- Bottle of 0.5M Sodium acetate
- Bottle of 0.5M acetic acid.
Goal: Obtain a pH of 5.
- Obtain a 50mL beaker or Conical tube, and add 10mL of 0.5M Sodium acetate
- Calculate the theoretical volume of 0.5M acetic acid required to obtain a pH 5 to be mixed in with the 10 mL of 0.5M Sodium acetate.
- Mix the calculated volume and submit the buffer to your instructor.

Evaluation: The percent error from your pH will be deducted from the total score.

If the percent error is 5%, then the total score will be 15/20.

pH	Student's pH measurements	Percent error (%)
5

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Post-Lab Questions

Summarize the key observations you made during the experiment. How did enzyme activity change with different pH levels?
How did calibration of the pH meter impact the accuracy of your measurements? What would happen if the meter were not calibrated properly?
Explain the buffering capacity observed in your experiment. Did the buffer effectively resist changes in pH? Why or why not?
Based on your results, how do extreme pH levels impact enzymatic function? What molecular interactions are responsible for these effects?
Reflect on the importance of the Henderson-Hasselbalch equation in predicting and controlling pH in biological systems. How did your experimental results compare to your calculated predictions?

Data Analysis

Compare your experimental data to your pre-lab predictions. Were your expectations met? Why or why not?
Discuss potential sources of error in your experiment and how they may have influenced your results.
How might the concepts from this lab apply to real-world biological or medical scenarios?

Learning Objectives

Pre-Lab Questions

Note

Post-Lab Questions

Data Analysis

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