Question

REFLECT AND APPLY Sephadex \(G-75\) has an exclusion limit of 80,000 molecular weight for globular proteins. If you tried to use this column material to separate alcohol dehydrogenase (MW 150,000 ) from \(\beta\) -amylase \((\mathrm{MW} 200,000),\) what would happen?

Short answer

Both proteins will elute together and won't be separated.

Step-by-step solution

Understand the Exclusion Limit

Sephadex \(G-75\) has an exclusion limit of 80,000 molecular weight (MW), which means it cannot separate molecules larger than 80,000 MW effectively.

Identify Protein Molecular Weights

Alcohol dehydrogenase has a molecular weight (MW) of 150,000, and \(\beta\)-amylase has a MW of 200,000. Both are larger than the exclusion limit of Sephadex \(G-75\).

Predict the Separation Outcome

Since both alcohol dehydrogenase and \(\beta\)-amylase exceed the exclusion limit of Sephadex \(G-75\), neither protein will be effectively separated. Instead, they will both elute in the void volume, essentially together.

Key concepts

Sephadex G-75

Sephadex G-75 is a type of gel filtration (or size-exclusion) chromatography medium. It is commonly used in laboratories to separate molecules based on their size. The medium consists of cross-linked dextran, which forms a gel with pores of a specific size. As molecules pass through the gel, smaller ones enter the pores and take longer to pass through, while larger molecules are excluded and move more quickly. Sephadex G-75 is particularly designed for separating molecules with molecular weights ranging up to 80,000 Daltons (Da).

Exclusion Limit

The exclusion limit in gel filtration chromatography refers to the maximum size of molecules that can enter the pores of the gel matrix. For Sephadex G-75, the exclusion limit is 80,000 molecular weight (MW). This means that any molecule larger than 80,000 MW will not be able to penetrate the pores of the gel and will elute from the column without any significant delay.

In other words, molecules above this size will pass through the column rapidly, as they are 'excluded' from entering the inner spaces of the gel. Conversely, smaller molecules will linger within the column longer because they can enter and exit the pores, causing them to travel more convoluted paths.

Protein Separation

Gel filtration chromatography is often used to separate proteins based on their size. In the context of Sephadex G-75, proteins larger than the exclusion limit of 80,000 MW will not be separated effectively. For instance, alcohol dehydrogenase (MW 150,000) and β-amylase (MW 200,000) are both significantly larger than 80,000 MW.

Consequently, when these proteins are passed through a column packed with Sephadex G-75, they will bypass the pores and elute together in the void volume. This makes Sephadex G-75 unsuitable for separating these particular proteins.

Understanding the characteristics of the chromatography medium and the molecular size of the proteins in question is vital for choosing the appropriate separation method. Effective protein separation often requires selecting a gel filtration medium with an exclusion limit that suits the molecular weights of the target proteins.