Protein Chemistry. Structure, Properties, Research Methods - Shendryk A. N. 2022

Protein Structure
Protein Structure
Determination of the Amino Acid Composition of Proteins

The Amino Acid Sequence of a polypeptide or protein constitutes its Primary Structure. Determining The amino acid sequence involves the following stages:

> Determination of the total Amino Acid Composition (gross composition);

> identification of the N- and C-terminal Amino Acids;

> fragmentation (Cleavage) of large protein peptide chains into smaller fragments containing fewer amino acid residues (20 - 60);

> determination of the amino acid sequence in short peptide fragments using Edman Degradation;

> Preparation of Peptide maps;

> final elucidation of the complete amino acid sequence of the peptide chain based on the results of all the aforementioned stages.

The Analysis of the total (gross) Amino acid composition of Proteins comprises two main steps: complete (nonspecific) Protein Hydrolysis and quantitative analysis of the resulting amino acid mixture.

Complete hydrolysis is carried out in Glass ampoules that are pre-cleaned with a hot mixture of sulfuric and nitric acids (3:1 by volume) for 10-15 hours. Following acid Treatment, the ampoules are rinsed with distilled deionized Water and dried in an air oven at 110 ∘C.

The standard method of acid hydrolysis (complete Protein Cleavage) is as follows: 1-5 mg of protein is placed in a glass ampoule, treated with 6 N HCl, evacuated, filled with nitrogen, sealed, and incubated at 110 ∘C for 12 - 36 hours.

The HCl solution is prepared as follows. Gaseous HCl is bubbled through water to obtain a 12 N Hydrochloric acid solution. This solution is then mixed with water in a 9:11 ratio and distilled. Distillation yields a 5.7 N HCl solution.

Protein cleavage during acid hydrolysis proceeds practically without Amino Acid Racemization. A major challenge is the complete destruction of the indole moiety of Tryptophan if it is present in the protein. Therefore, to avoid these complications, methanesulfonic acid can be used instead of HCl, as it does not destroy tryptophan.

During acid hydrolysis, the amides glutamine and asparagine are converted into ammonia and glutamic and aspartic acids, respectively. This conversion is detected and accounted for by measuring The amount of ammonia released and utilizing sequencing data.

Serine, Threonine, Cysteine, Tyrosine, and Methionine are partially degraded during acid hydrolysis. Furthermore, peptide bonds involving Val, Leu, and Ile undergo hydrolysis with difficulty and incompletely. To account for these effects, The kinetics of hydrolysis is studied by plotting the concentration of the aforementioned amino acids against time. Based on the resulting graphs, the amounts of partially degraded Amino acids are determined by extrapolation to zero time, while those of the poorly hydrolyzed ones are found when the curve reaches a plateau.

This is qualitatively illustrated in Fig.

Upon completion of hydrolysis, hydrochloric acid is removed from the ampoule by evaporation. In addition to acid hydrolysis, alkaline hydrolysis is also employed. In this case, one must account for the following complications: the Degradation of cysteine, cystine, serine, and threonine. Furthermore, alkaline hydrolysis leads to the racemization of all amino acids.

Class="center">

Analysis of amino acid mixtures following hydrolysis

The Quantitative determination of amino acids obtained after nonspecific Hydrolysis of Proteins and Peptides is typically carried out using various partition Chromatography Methods. Some of the simplest Examples are outlined below.

Thin-Layer Chromatography is the simplest and most accessible method for separating amino acids. Its quantitative evaluation capabilities are limited and labor-intensive. As a rule, strictly quantitative analysis is not performed after Separation in a thin layer; only approximate estimates (greater than, less than, etc.) are provided when possible. Identification (assignment) of developed amino acid spots is carried out by comparing chromatograms with those of standard amino acid mixtures of known composition run under identical conditions.

Column chromatography. Column Ion-exchange chromatography is the most common method used for amino acid separation. The stationary phase consists of sulfonated polystyrene cross-linked with divinylbenzene. In this chromatography technique, amino acids are converted into their cationic form at low pH values, allowing them to bind to the charged sulfo groups of the support. Elution is performed using a buffer (such as sodium citrate) with a gradually increasing pH. Raising the pH reduces the effective positive charges of the Amino Acids and, consequently, the strength of their binding to the resin. This achieves the sequential (stepwise) displacement (elution) of amino acids from the column. The order in which amino acids elute from the column is largely determined by the pH of the eluting buffer. During the elution process, Na+ ions displace the amino acids from their bonds with the sulfo groups.

A two-column Procedure is also known. The first column, packed with Amberlite IR-120 for instance, is used to separate acidic and neutral amino acids, while the analysis of basic amino acids is performed on a second column. This technique has not found widespread application because it is technically rather complex to implement. In addition, it approximately doubles the consumption of analytical Materials (due to losses from irreversible binding) and significantly increases measurement error.

As mentioned earlier, the pH of the buffer mixture must increase during elution. This gradient can be either continuous or discontinuous; the latter is much simpler and therefore more widely used.

Internal standard. A precisely known amount of a specific amino acid is added to the protein solution as an internal standard prior to hydrolysis. The quantitative recovery metrics of this standard from the column serve as a baseline for calculating the yields of all amino acids obtained during hydrolysis. This approach accounts for any amino acid losses incurred during the analytical procedure.

Detection of amino acids eluting from the column and determination of their structure. As noted above, Proteinogenic Amino Acids are colorless compounds, making it impossible to visually track their movement along the column and at its outlet. To accomplish this, a UV detector must be used, or the amino acids must be "stained" or chemically modified. Such derivatization is performed in two ways.

1. Post-column derivatization. Prior to the 1970s, amino acids eluted from the column were detected using the ninhydrin reaction. The intensity of the resulting color was measured at two wavelengths: initially at 580нм and subsequently at 440нм to determine the content of Proline and hydroxyproline.

In 1972, fluorescamine was introduced as a selective fluorogenic reagent for primary amines. It provides a sensitivity 10 to 100 times greater than the ninhydrin method. Measurements are performed using a fluorimeter with an excitation wavelength of 340 нм and an emission wavelength of 450 нм (Stokes shift of the band).

Fluorescamine is an extremely expensive compound that is unstable in aqueous media, and therefore it is rarely used. It can be—and successfully is—replaced by o-phthalaldehyde (OPA). The fluorescent OPA-amino acid derivative exhibits an excitation band with a maximum absorption wavelength of 360 нм and an emission wavelength of 455 нм. This derivative readily forms at pH=9-11. Certain complications in amino acid detection with OPA arise during the analysis of cysteine, cystine, proline, and hydroxyproline. The sensitivity of amino acid determination using OPA is 5пмоль (for comparison, with ninhydrin it is 100 пмоль).

2. Pre-column derivatization. Post-column amino acid derivatization is a complex and labor-intensive procedure. It requires periodically sampling aliquots of the eluent, treating them with appropriate Reagents, and analyzing them via photocolorimetry or fluorometry. It is simpler to perform pre-column derivatization of all amino acids immediately following hydrolysis, and then separate (analyze) the resulting mixture of modified amino acids using thin-layer, column, liquid, high-performance liquid (HPLC), or other chromatography methods. In terms of reproducibility, this approach is somewhat inferior to post-column derivatization. The most widespread method of pre-column derivatization is The conversion of amino acids into phenylthiohydantoin derivatives via the Edman reaction (see Section 1.1.6).

To facilitate and streamline high-throughput (routine) amino acid analyses, automated amino acid analyzers have been developed and manufactured by the instrument-making industry. These are highly sophisticated instruments based on the principles of High-Performance Liquid Chromatography.

Gas-Liquid Chromatography (GLC). In this variant of chromatographic analysis for amino acid mixtures, the amino acids are converted into volatile derivatives. A well-known single-step modification method is trimethylsilylation of NH₂, COOH, OH, and SH groups. Disadvantage: Certain amino acids yield multiplet peaks during chromatography.

Two-step modification protocols are more successful. Step 1 involves the Esterification of carboxyl groups. Step 2 involves the acylation of other reactive functional amino, sulfhydryl, hydroxyl, and guanidino groups.

While the GLC method provides satisfactory qualitative results, obtaining accurate quantitative data is extremely difficult. Furthermore, complications arise in determining Histidine, Arginine, and cystine, as the latter compounds are degraded on certain stationary phases. With the advent of automated amino acid analyzers, GLC has lost its significance and is employed less and less frequently.

Complete nonspecific hydrolysis of a protein (peptide) only establishes its gross composition, i.e., which amino acids comprise the peptide chain and in what quantitative ratios (proportions). However, this result yields no information regarding the sequence in which these amino acids are linked together. To solve this problem, a procedure known as peptide chain sequencing is performed. An integral part of this process is the determination of The structure of the N- and C-terminal amino acids.