Amino Acid Hydrolysis - Online Tutor, Practice Problems & Exam Prep

Amino acid hydrolysis involves the cleavage of peptide bonds using water, often accelerated by an acid catalyst like hydrochloric acid (6 M). This process releases free amino acids, which can be quantified using Ninhydrin, producing a bluish-purple color measurable by spectrophotometry. While amino acid composition can be analyzed through techniques like HPLC, the sequence of amino acids is lost during hydrolysis. Understanding this technique is crucial for protein analysis in proteomics, emphasizing the distinction between composition and sequence in protein structure.

Amino Acid Hydrolysis

In this video, we're going to talk about our first protein cleavage technique, amino acid hydrolysis. So recall that way back in our previous lessons when we talked about peptide bonds, we said that hydrolysis is just a reaction in which bonds are cleaved with the treatment of water. And so amino acid hydrolysis is specifically when peptide bonds are cleaved with the treatment of water. In our example below, we're going to look at the hydrolysis of a peptide bond. What you'll notice on the far left is a dipeptide or a peptide with just 2 amino acid residues. These 2 amino acid residues are linked via a peptide bond, shown in pink. We know that if we treat our dipeptide with water, we can induce hydrolysis. But we also know from our previous lesson videos that hydrolysis is a very slow reaction. To speed up this reaction, we can add an acid catalyst, represented here by H⁺. Our dipeptide, upon hydrolysis, ends up forming 2 free amino acids: one free amino acid on the far left, and another on the right. Complete amino acid hydrolysis can be induced with 6 molar hydrochloric acid, which non-specifically cleaves all the peptide bonds. This results in all the constituent amino acids of the protein being released as free amino acids.

Again, observe that we have a protein on the far left, with a bunch of amino acids represented by these circles. We have a free amino group on the N-terminal end of our protein and a free carboxyl group on the C-terminal end. When we treat our protein with 6 molar hydrochloric acid, we can induce complete amino acid hydrolysis, where all peptide bonds with the presence of water are cleaved. Most of our proteins are in an aqueous solution that contains water, leading to the cleavage of all peptide bonds and the release of all constituent free amino acids. These amino acids are no longer linked together by peptide bonds. A chemical known as Ninhydrin reacts with these free amino acids to produce a color for quantification. This allows us to quantify the number of amino acids via light absorbance using a spectrophotometer.

This chemical, known as Ninhydrin, reacts with the free amino acids to produce a bluish-purple color quantified with light absorbance using a spectrophotometer. With this data, we can analyze the amino acids using techniques such as mass spectrometry or HPLC to determine the protein or amino acid composition. Analyzing these amino acids with HPLC reveals the amino acid composition of the protein. It is important to note that once we use 6 molar hydrochloric acid, we cannot determine the sequence of our protein. Although we can determine the composition, we no longer know the exact order of amino acids from the N-terminal to the C-terminal end. However, we can readily identify the composition, which is crucial to keep in mind. This concludes our lesson on amino acid hydrolysis, and we'll practice utilizing these concepts in our next video. I'll see you there.

What is the function of ninhydrin?

• A. Cleavage of proteins into free amino acids.
• B. Colorimetric agent to detect amino acids.
• C. Agent to carboxymethylate cysteines.
• D. Serves as the mobile phase in HPLC.

Amino acid hydrolysis via 6M HCl cleaves all the amide/peptide bonds of a protein. What do you suppose happens to the amide bonds in the R-groups of Asn & Gln residues upon treatment with 6M HCl?

So this practice problem says, amino acid hydrolysis via 6 molar hydrochloric acid cleaves all of the amide/peptide bonds of a protein. It then asks, what do you suppose happens to the amide bonds in the R groups of both asparagine and glutamine residues upon treatment with 6 molar hydrochloric acid. Below, I've drawn the structure of the asparagine amino acid on the left as well as the glutamine amino acid on the right. You'll notice that in the R groups of both asparagine and glutamine, there is indeed an amide bond that is present and is highlighted in green here. We know that upon exposure to 6 molar hydrochloric acid, all of the amide and peptide bonds of a protein are going to be cleaved. That includes the amide bonds that are found in the R groups of both asparagine and glutamine. Starting with asparagine on the left, we know that upon exposure to 6 molar hydrochloric acid, the amide bond in the R group is going to be cleaved. This will result in the same exact R group, except instead of having the amino group at the end, there will be an oxygen atom at the end. The resulting amino acid is aspartic acid, whose three-letter code is ASP and one letter code is D. This indicates that asparagine residues can be converted into aspartic acid residues upon hydrolysis. Applying the same logic to glutamine on the right, we know upon exposure to 6 molar hydrochloric acid, the amide bond in glutamine’s R group will be cleaved. This results in the same exact R group, except instead of having the amino group at the end, there will be an oxygen atom and a carboxylate group. This R group is essentially the R group of glutamic acid. Glutamic acid's three-letter code is GLU, and its one-letter code is E. Thus, upon 6 molar hydrochloric acid exposure, glutamine residues can be converted into glutamic acid residues. What this practice problem is hinting at is that even though we know amino acid hydrolysis can be used to determine the amino acid composition of a protein, it does not always allow for complete amino acid analysis. This is partly because asparagine residues can be converted to aspartic acid, and glutamine residues can be converted to glutamic acid. Thus, we also have several different protein cleavage techniques to determine the amino acid composition and sequence of a protein, such as the use of peptidases, which we will talk more about later in our course. This concludes our practice problem, and I'll see you guys in our next video.

Here’s what students ask on this topic:

What is amino acid hydrolysis and how does it work?

Amino acid hydrolysis is a process where peptide bonds in proteins are cleaved using water, often accelerated by an acid catalyst like 6 M hydrochloric acid (HCl). This reaction breaks down proteins into their constituent free amino acids. The hydrolysis reaction is generally slow, but the addition of an acid catalyst speeds it up. The process involves treating the protein with water and HCl, which cleaves all peptide bonds, releasing free amino acids. These free amino acids can then be quantified and analyzed using techniques like Ninhydrin reaction and HPLC.

Why is 6 M hydrochloric acid used in amino acid hydrolysis?

6 M hydrochloric acid (HCl) is used in amino acid hydrolysis because it acts as a strong acid catalyst that significantly speeds up the hydrolysis reaction. Without the acid, the reaction would be very slow. The 6 M HCl ensures complete hydrolysis of all peptide bonds in the protein, releasing all constituent amino acids as free amino acids. This complete hydrolysis is crucial for subsequent analysis of amino acid composition using techniques like Ninhydrin reaction and HPLC.

How does Ninhydrin help in quantifying amino acids after hydrolysis?

Ninhydrin is a chemical reagent that reacts with free amino acids to produce a bluish-purple color. After amino acid hydrolysis, the free amino acids are treated with Ninhydrin, which forms a colored complex. This color can be quantified using a spectrophotometer, which measures light absorbance. The intensity of the color is directly proportional to the concentration of amino acids, allowing for accurate quantification. This method is essential for determining the number of amino acids present in a hydrolyzed protein sample.

What are the limitations of using 6 M hydrochloric acid in amino acid hydrolysis?

While 6 M hydrochloric acid (HCl) is effective for complete hydrolysis of peptide bonds, it has limitations. One major limitation is that it destroys the sequence information of the protein. The hydrolysis process breaks all peptide bonds, making it impossible to determine the exact order of amino acids (sequence) from the N-terminal to the C-terminal end. Therefore, while the composition (types and number of amino acids) can be determined, the sequence information is lost. This is a critical consideration in proteomics where sequence information is often required.

What techniques are used to analyze amino acid composition after hydrolysis?

After amino acid hydrolysis, the free amino acids can be analyzed using techniques such as High-Performance Liquid Chromatography (HPLC) and mass spectrometry. HPLC separates amino acids based on their chemical properties, allowing for the identification and quantification of each amino acid in the sample. Mass spectrometry provides detailed information about the molecular weight and structure of the amino acids. These techniques are essential for determining the amino acid composition of proteins, although they do not provide sequence information.