Question

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 (MW 200,000 ), what would happen?

Short answer

Both proteins will elute together and cannot be separated using Sephadex G-75 due to their large molecular weights.

Step-by-step solution

Understand the Exclusion Limit

The exclusion limit of Sephadex G-75 is 80,000 molecular weight (MW). This means that any molecules larger than 80,000 MW will not enter the pores of the gel beads and will elute (come out) first from the column.

Identify the Molecular Weights

Identify the molecular weights of the proteins to be separated. Alcohol dehydrogenase has a molecular weight of 150,000 and \( \beta \)-amylase has a molecular weight of 200,000.

Determine the Relation to Exclusion Limit

Both alcohol dehydrogenase (150,000 MW) and \( \beta \)-amylase (200,000 MW) are above the exclusion limit of 80,000 MW. Therefore, both proteins will be too large to enter the beads and will elute from the column together.

Predict the Separation Outcome

Since both proteins exceed the molecular weight exclusion limit of the Sephadex G-75, they will not be separated by the column material. Instead, they will both be eluted in the void volume.

Key concepts

Sephadex G-75

Sephadex G-75 is a type of size exclusion chromatography gel. It is commonly used in laboratories for separating molecules based on their size. The 'G-75' indicates the specific type of Sephadex with a certain pore size. Sephadex is made of dextran, a polysaccharide, which forms a gel matrix.
The gel beads of Sephadex G-75 have a specific pore size, which determines the exclusion limit. Due to the gel's porous nature, molecules larger than a certain size cannot enter the pores and are eluted first. Smaller molecules can enter the pores and elute later.
So, Sephadex G-75 is a useful tool in biochemical research when separating molecules such as proteins, peptides, and polysaccharides based on their size.

Exclusion Limit

The exclusion limit is a critical concept in size exclusion chromatography. For Sephadex G-75, the exclusion limit is 80,000 molecular weight (MW). This means any molecules larger than 80,000 MW cannot enter the pores of the gel beads.
When a mixture is applied to a Sephadex G-75 column, larger molecules will be excluded from the beads and will elute from the column first. These molecules travel through the column without entering the pores. Smaller molecules, which can enter the pores, will elute later.
Understanding the exclusion limit is crucial for determining which molecules can be separated using a specific type of Sephadex gel. For example, if you want to separate two proteins that both have MWs above 80,000, they will elute together and will not be separated by Sephadex G-75.

Protein Separation

Protein separation involves distinguishing proteins based on their size, charge, or affinity to certain substances. Size exclusion chromatography, such as using Sephadex G-75, separates proteins based on their size.
When separating proteins, it's essential to know their molecular weights relative to the exclusion limit of the gel. If proteins are larger than the exclusion limit, like alcohol dehydrogenase (MW 150,000) and \( \beta \)-amylase (MW 200,000), they will not be separated by the column material since both exceed 80,000 MW.
To successfully separate proteins using size exclusion chromatography, the proteins should have different sizes within the gel's effective range. This allows proteins to move at different speeds through the column, enabling separation.

Molecular Weight

Molecular weight (MW) is a measure of the mass of a molecule. It is usually expressed in Daltons (Da) or unified atomic mass units (u). In protein chemistry, understanding the MW of proteins helps in selecting the right type of chromatography for separation.
In the context of Sephadex G-75, knowing that alcohol dehydrogenase has a MW of 150,000 and \( \beta \)-amylase has a MW of 200,000 helps us predict their behavior in the gel. Since both proteins are significantly larger than the exclusion limit of 80,000 MW, they will both elute together in the void volume.
When planning an experiment involving protein separation, it's crucial to gather accurate MW information for all proteins involved. This allows for better decision-making regarding the appropriate method and materials for effective separation.