Although there are many methods for analyzing the amino acid composition of peptide synthesis , and tend to be more sensitive, more precise, simpler and faster, no single method can be applied to the detection of all amino acid residues alone, and many factors such as temperature, time, Hydrolysis reagents, hydrolysis methods, and additives in peptide synthesis samples all have an effect on the degree of hydrolysis. The commonly used hydrolysis methods are briefly described as follows:
1 acidic hydrolysis
Acidic hydrolysis is the most widely used hydrolysis method, allowing most amino acid residues to be completely hydrolyzed. The most common hydrolyzing agent is 6 mol/L HCI.
Conditions: 6 mol/L HCI, vacuum, 110 ° C. The hydrolysis time is 20-24 h. It can be used in both liquid phase hydrolysis mode and gas phase hydrolysis mode. However, under this condition, asparagine and glutamine were completely hydrolyzed to aspartic acid and glutamic acid, respectively, and tryptophan was completely destroyed. Cysteine ​​was firstly dithiodipropionic acid, 4 Hydrolysis of ethylene acridine or iodoacetic acid can be determined by hydrolysis, and the tyrosine moiety is destroyed by the hydrolyzate, and serine and threonine are partially hydrolyzed. The peptide bond between some aliphatic amino acid residues is difficult to cleave and can be solved by prolonging the hydrolysis time such as hydrolysis for 92h or even 120h.
2 alkaline hydrolysis
Alkaline hydrolysis generally uses NaOH and KOH as hydrolyzing agents. This hydrolysis method is a complementary method of HCI hydrolysis. Because most amino acids are destroyed or racemized during base hydrolysis, only tryptophan is stable. So this method is limited to the determination of tryptophan.
3 enzyme hydrolysis
The hydrolysis of peptide chains with a set of proteases is particularly suitable for the determination of chemically susceptible amino acids such as asparagine and glutamine. The amino acid does not undergo racemization during the hydrolysis process, and almost all of the constituent amino acids are not destroyed. However, because the enzymatic hydrolysis conditions are mild, there is no destructive effect on asparagine, glutamine, etc., and the reaction takes a long time, the hydrolysis is incomplete, and the enzyme itself is a protein, which may interfere with the measurement results of the sample.
Microwave radiation can be hydrolyzed. Under normal conditions, the peptide can be completely hydrolyzed by microwave hydrolysis at 190 ° C for 15 minutes, resulting in a much shorter time for complete hydrolysis. In microwave-assisted acid hydrolysis and microwave-assisted enzymatic hydrolysis, the hydrolysis time can be shortened from tens of hours to tens of minutes in the past.
In summary, the hydrolysis method of the peptide synthesis drug should be carried out according to the nature of the product, the purpose of the study, the amount of sample available, and the specific conditions of the laboratory.
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