Angiotensins I, II and III are physiologically important peptides for blood pressure control* and they contain an arginyl residue in their molecules. Renin, an endopeptidase, releases angiotensin I from endogenous angiotensinogen. Angiotensin II, one of most potent vasoconstrictors known, is generated from angiotensin I by angiotensin-converting enzyme. In addition, an aminopeptidase removes the amino-terminal aspartic acid residue from angiotensin II yielding angiotensin III.
These peptides have generally been measured by radioimmunoassay (RIA), since these methods are sensitive enough to permit the determination of as little as 510 fmole amounts of the peptides 24. However, high-performance liquid chromatography (HPLC) coupled with highly sensitive detection may be more suitable for either determination of angiotensins or for a study of the enzyme kinetics in the renin-angiotensin system, because the HPLC method permits simultaneous determination of angiotensins I, II and III and other related peptides. Although conventional peptide analyses with HPLC are generally performed by ultraviolet (W) absorption detection at wavelengths between 200 and 230 nm5-', post-column fluorescence detections-10 or electrochemical detection' I, the sensitivities of the HPLC methods are still three to four orders of magnitude lower than those of the RIA methods.
We have developed recently a pre-column fluorescence derivatization method in HPLC for guanidino compounds, such as methylguanidine and guanidinosuccinic acid, by using a fluorogenic reagent, benzoin 12. Renzoin reacts with the guanidino moiety of compounds in an alkaline medium, in the presence of 2-mercaptoethanol (to stabilize the fluorescent product) and sodium sulphite (to suppress blank fluorescence)* 3, and it yields highly fluorescent derivatives, viz. 2-substituted amino-4,5-diphenylimidazoles14. Therefore, it is conceivable that arginine-containing peptides are also converted into the corresponding fluorescent derivatives by the benzoin reaction.
This paper describes a simple and sensitive HPLC method by means of the pre-column derivatization with benzoin for the simultaneous determination of angiotensins I, II and III. The HPLC method allows the quantitative determination of the peptides at the femtomole level. In the investigations, four other arginine-contaming peptides, namely, tuftsin, substance P, luteinizing hormone-releasing hormone (LH-RH) and leupeptin acid were also examined with regard to the derivatization conditions.