Techniques for analyzing protein content, purity, molecular weight, amino acid composition and structure, etc.
Content analysis, amino acids are nitrogen-containing compounds, general protein contains about 15% nitrogen, the Kjeldahl method can be used to calculate the protein content in the sample. The amount of peptide chain was measured by the biuret method. The absorption value at the wavelength of 280nm was measured by ultraviolet spectrophotometer, that is, the value of aromatic amino acid in the protein. The content of protein can be calculated by methods such as measuring the value of lysine in protein by using benzenesulfonic acid). Simple proteins contain only amino acids and are easy to measure. Complex proteins also contain prosthetic groups, such as sugars, lipids, nucleic acids and metals, and other chemical substances, which need to be identified by other analytical methods.
Amino acids are nitrogen-containing compounds. Generally, proteins contain about 15% nitrogen. The protein content in the sample can be calculated by the Kjeldahl method. The amount of peptide chain was measured by the biuret method. The absorption value at the wavelength of 280nm was measured by ultraviolet spectrophotometer, that is, the value of aromatic amino acid in the protein. The content of protein can be calculated by methods such as measuring the value of lysine in protein by using benzenesulfonic acid). Simple proteins contain only amino acids and are easy to measure. Complex proteins also contain prosthetic groups, such as sugars, lipids, nucleic acids and metals, and other chemical substances, which need to be identified by other analytical methods.
The protein is purified from the sample, and its purity identification methods include polyacrylamide gel electrophoresis, electrofocusing electrophoresis and end analysis. The protein was considered purified if both electropherograms exhibited a single band or constant terminal amino acids.
Molecular weight determination
Generally, those with a molecular weight greater than 10,000 are called proteins, and those with a molecular weight of less than 10,000 are called polypeptides. The largest proteins can have molecular weights in the millions. Determination methods such as gel filtration, SDS-polyacrylamide gel electrophoresis and ultracentrifugation, etc., which are used to determine proteins with larger molecular weights, require sophisticated and expensive instruments.
Amino acid composition analysis
After acid, alkali or enzyme treatment, the peptide chain of the protein is disassembled, that is, hydrolyzed into free amino acids. The 20 amino acids that make up the protein can be qualitatively identified by paper chromatography and thin layer chromatography. The content of various amino acids can be accurately determined by ion exchange chromatography. The amino acid automatic analyzer designed and manufactured based on the principle of ion exchange chromatography can accurately and quantitatively determine the amino acid composition of proteins, and is a widely used method at present.
Each protein has its own specific amino acid sequence of polypeptide chains, ie, primary structure. Polypeptide chains in turn form spatial structures in a specific coiled and folded manner, namely secondary, tertiary and quaternary structures.
The main method for determining the primary structure: according to the chemical reaction invented by Edman, the reagent phenyl isothiocyanate is degraded into amino acid derivatives one by one from the N (-NH2) end of the chain, and each step of the reaction is identified. , the amino acid sequence of the peptide chain can be derived. The protein sequencer designed according to this principle can automatically determine 40 to 50 sequences at a time with a sample of milligrams or micrograms. If the complete primary structure is to be obtained, the protein still needs to be cleaved into fragments of the peptide, after the separation and purification of the peptide, the sequence of the peptide is measured and then connected into a complete sequence. Despite sophisticated automated instrumentation, primary structural analysis is a complex, high-volume, and time-consuming task.
In recent years, the method of determining the sequence of DNA and then deducing the amino acid sequence has been widely used, which greatly simplifies the sequence analysis. Spatial structure determination includes the use of crystal X-diffraction analysis, CD spectroscopy, nuclear magnetic resonance spectroscopy (NMR), and laser Raman spectroscopy, and requires more sophisticated techniques and expensive instruments.
Intact Protein Analysis