91Ó°ÊÓ

Ferritin is a hollow iron-storage protein \({ }^{61}\) consisting of 24 subunits that are a variable mixture of heavy \((\mathrm{H})\) or light \((\mathrm{L})\) chains, arranged in octahedral symmetry. The hollow center, with a diameter of \(8 \mathrm{~nm}\), can hold up to 4500 iron atoms in the approximate form of the mineral ferrihydrite \(\left(5 \mathrm{Fe}_{2} \mathrm{O}_{3} \cdot 9 \mathrm{H}_{2} \mathrm{O}\right)\). Iron(II) enters the protein through eight pores located on the threefold symmetry axes of the octahedron. Oxidation to Fe(III) occurs at catalytic sites on the \(\mathrm{H}\) chains. Other sites on the inside of the \(\mathrm{L}\) chains appear to nucleate the crystallization of ferrihydrite. Migration times for protein standards and the ferritin subunits are given in the table. Prepare a graph of log(molecular mass) versus \(1 /\) (relative migration time), where relative migration time \(=\) (migration time)/(migration time of marker dye). Compute the molecular mass of the ferritin light and heavy chains. The masses of the chains, computed from amino acid sequences, are 19766 and \(21099 \mathrm{Da}\). $$ \begin{array}{lcl} \text { Protein } & \begin{array}{c} \text { Molecular } \\ \text { mass (Da) } \end{array} & \begin{array}{l} \text { Migration } \\ \text { time (min) } \end{array} \\ \hline \text { Orange G marker dye } & \text { small } & 13.17 \\ \alpha \text {-Lactalbumin } & 14200 & 16.46 \\ \text { Carbonic anhydrase } & 29000 & 18.66 \\ \text { Ovalbumin } & 45000 & 20.16 \\ \text { Bovine serum albumin } & 66000 & 22.36 \\ \text { Phosphorylase B } & 97000 & 23.56 \\ \beta \text {-Galactosidase } & 116000 & 24.97 \\ \text { Myosin } & 205000 & 28.25 \\ \text { Ferritin light chain } & & 17.07 \\ \text { Ferritin heavy chain } & & 17.97 \\ \hline \end{array} $$

Step by step solution

01

Calculate Relative Migration Times

To begin, calculate the relative migration time for each protein by dividing the migration time of each protein by the migration time of the Orange G marker dye (13.17 min). This yields the following relative migration times: \(\alpha\)-Lactalbumin: \( \frac{16.46}{13.17} \approx 1.25 \), Carbonic anhydrase: \( \frac{18.66}{13.17} \approx 1.42 \), Ovalbumin: \( \frac{20.16}{13.17} \approx 1.53 \), Bovine serum albumin: \( \frac{22.36}{13.17} \approx 1.70 \), Phosphorylase B: \( \frac{23.56}{13.17} \approx 1.79 \), \(\beta\)-Galactosidase: \( \frac{24.97}{13.17} \approx 1.90 \), Myosin: \( \frac{28.25}{13.17} \approx 2.14 \), Ferritin light chain: \( \frac{17.07}{13.17} \approx 1.30 \), Ferritin heavy chain: \( \frac{17.97}{13.17} \approx 1.36 \).

02

Calculate Logarithm of Molecular Masses

Next, calculate the logarithm (base 10) of the molecular masses given for each standard protein. For example, for \( \alpha\)-Lactalbumin (14200 Da), log(molecular mass) is \( \log_{10}(14200) \approx 4.15 \). Repeat for each protein: Carbonic anhydrase (29000 Da): \( \log_{10}(29000) \approx 4.46 \), Ovalbumin (45000 Da): \( \log_{10}(45000) \approx 4.65 \), Bovine serum albumin (66000 Da): \( \log_{10}(66000) \approx 4.82 \), Phosphorylase B (97000 Da): \( \log_{10}(97000) \approx 4.99 \), \( \beta\)-Galactosidase (116000 Da): \( \log_{10}(116000) \approx 5.06 \), Myosin (205000 Da): \( \log_{10}(205000) \approx 5.31 \).

03

Graphical Representation

Plot the calculated log(molecular mass) on the y-axis versus \(1 /\text{(relative migration time)}\) on the x-axis for the protein standards. This allows us to establish a calibration curve.

04

Determine Equation of Best Fit

Using the plot from Step 3, draw the best line of fit through the points. The relationship should appear linear. Use this line to establish an equation in the form \( y = mx + c \), where \( y \) is the log(molecular mass) and \( x \) is \( 1/\text{(relative migration time)} \).

05

Calculate Molecular Masses of Ferritin Chains

For each of the ferritin chains' relative migration times, calculate \(1 / \text{(relative migration time)}\) and use it in the equation derived in Step 4 to solve for log(molecular mass). Convert this back to the molecular mass by taking the antilog (base 10). For the light chain with a relative migration time of 1.30, calculate \( \frac{1}{1.30} \approx 0.769 \). Similarly, for the heavy chain with a relative migration time of 1.36, calculate \( \frac{1}{1.36} \approx 0.735 \). Apply these values to the equation for approximate molecular mass.

Unlock Step-by-Step Solutions & Ace Your Exams!

• Full Textbook Solutions

  Get detailed explanations and key concepts

• Unlimited Al creation

  Al flashcards, explanations, exams and more...

• Ads-free access

  To over 500 millions flashcards

• Money-back guarantee

  We refund you if you fail your exam.

Over 30 million students worldwide already upgrade their learning with 91Ó°ÊÓ!

Key Concepts

These are the key concepts you need to understand to accurately answer the question.

Molecular Mass Determination

Understanding how to determine the molecular mass of proteins like the ferritin subunits is crucial in biochemistry. We use a technique called protein electrophoresis, which allows us to analyze proteins based on their size by observing their migration through a gel under an electric field. The molecular mass determination can be achieved by comparing unknown proteins to standards whose sizes are known. This method involves plotting a calibration curve using a graph of log(molecular mass) versus the reciprocal of relative migration time.

• Log of Molecular Mass: This is calculated by taking the logarithm (base 10) of the molecular weights of the known standard proteins.
• Relative Migration Time: Calculated by dividing the migration time of each protein by that of a marker dye, providing a relative metric to compare.

By using the linear relationship observed from the graph, we can interpolate or extrapolate to estimate the molecular mass of the ferritin subunits.

Iron-Storage Proteins

Ferritin exemplifies an iron-storage protein, playing a vital role in iron metabolism. It's a spherical complex made up of 24 subunits, capable of storing iron in a non-toxic form, and releasing it when needed. These subunits consist of heavy (H) and light (L) chains, contributing to ferritin's functionality.

• Iron Storage: Ferritin's core can store up to 4500 iron atoms arranged in the form of the mineral ferrihydrite.
• Structure: The protein has an octahedral symmetry with 8 pores that allow iron ions to enter.

Ferritin ensures regulation of iron levels in cells, preventing both deficiency and excess, which can lead to oxidative stress. Through its design and ability to encapsulate iron, ferritin helps maintain cellular health and function.

Catalytic Sites in Proteins

Catalytic sites are essential components of many proteins, enabling them to facilitate biochemical reactions. In ferritin, these sites are located on the heavy (H) chains of the protein. They are crucial for the oxidation of iron from its ferrous (Fe II) to its ferric (Fe III) form.

• Oxidation Process: Iron (II) entering ferritin is oxidized at these sites, crucial for forming the stable ferrihydrite mineral inside the protein.
• Nucleation: The light (L) chains help in nucleating ferrihydrite crystallization, a vital step in storing metal ions efficiently.

This oxidation not only stores iron securely but also aids in detoxifying the metal, preventing potential damage from iron's reactive forms.

Protein Migration Time Analysis

Analyzing protein migration time during electrophoresis provides insights into a protein's characteristics. The time it takes for a protein to move through the gel is influenced by its size and shape. By comparing migration times with those of known standards, the relative sizes of proteins can be accurately determined.

• Relative Time Calculation: This involves dividing the migration time of each protein by that of a standard marker dye, facilitating comparisons.
• Molecular Mass Estimation: The graph plotted as log(molecular mass) vs. reciprocal of the relative migration time helps in estimating unknown molecular weights.

Through this approach, one can deduce the relative molecular sizes of proteins, linking the physical observations during electrophoresis to biochemical properties such as the molecular mass of the ferritin subunits.