Page 13 - Application Handbook - Liquid Chromatography

Application No.L458
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■ Analysis of Angiotensin I Hydrolysate
Angi oten sin is a po ly pe ptid e tha t ex hi bi ts After adding 500 µL of 6 mol/L hydrochloric acid (ICP
vasoconstriction activity to increase blood pressure. grade) to 0.5 mg of angiotensin Ⅰ, the mixture was set
There are four types of angiotensin, types Ⅰ- Ⅳ, that aside for 22 hours in a reduced pressure atmosphere at
differ in the numbers and types of amino acids in their 110 °C to permit complete hydrolysis.
structures. The liquid phase was then evaporated under a stream
The structure of angiotensin Ⅰconsists of a total of ten of nitrogen gas to obtain a dry residue. The residue was

amino acid residues, including aspartic acid, arginine, then re-dissolved in 10 mL of 0.1 mol/L borate buffer
valine, tyrosine, isoleucine, proline, phenylalanine, for use as the sample.
leucine, and two molecules of histidine. In response to The analysis results are shown in Fig. 2. Two molecules
the action of angiotensin-converting enzyme (ACE), of histidine were detected while one molecule each of

angiotensin Ⅰ is converted to angiotensin Ⅱ, which the other amino acids was detected, which matched
exhibits greater vasopressor activity. the actual amino acid sequence.

mV
300
■ Peaks
250 1. Aspartic Acid, 2. Histidine, 3. Argininge, 4. Tyrosine, 5. Valine,
6. Phenylalanine, 7. Isoleusine, 8. Leucine, 9. Proline
200 9

150

100 1 2 3 8
4
6 7
50
5
0

0.0 2.5 5.0 7.5 min
Fig. 2 Chromatogram of Angiotensin I Hydrolysate

■ Analysis of Bovine Serum Albumin Hydrolysate
Bovine serum albumin is a protein that is often used in The liquid phase was then evaporated under a stream
biochemical experiments. In this example, after adding of nitrogen gas to obtain a dry residue. The residue was
500 µL of 6 mol/L hydrochloric acid (ICP grade) to then re-dissolved in 10 mL of 0.1 mol/L borate buffer
3.5 mg of bovine serum albumin, the mixture was for use as the sample. The results of analysis are shown
placed in a reduced pressure atmosphere at 110 °C for in Fig. 3.
22 hours to permit complete hydrolysis.

mV
400 ■ Peaks
350 1. Aspartic Acid, 2. Glutamic Acid, 3. Serine, 4. Histidine, 5. Glycine, 6. Threonine,
7. Argininge, 8. Alanine, 9. Tyrosine, 10. Valine, 11. Methionine,
300 12. Phenylalanine, 13. Isoleusine, 14. Leucine, 15. Lysine, 16. Proline
250 2 16
200 1 8 14
150
100 3 7 15
6 9 10 12
50 4 5 13
11
0
-50
0.0 2.5 5.0 7.5 min
Fig. 3 Chromatogram of Bovine Serum Albumine Hydrolysate

First Edition: Jul. 2014

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