Dear @Samyr Querobino

HPLC is extremely versatile for the isolation of peptides from a wide variety of synthetic or biological sources. The number of applications of HPLC in peptide and protein purification continue to expand at an extremely rapid rate. Solid-phase peptide synthesis and recombinant DNA techniques have allowed the production of large quantities of peptides which need to be highly purified. The design of multidimensional purification schemes to achieve high levels of product purity further highlight the power of HPLC techniques in the analysis and isolation of peptide samples. The complexity of the mixture to be chromatographed depends on the nature of the

source and the degree of preliminary clean-up that can be performed. In the case of synthetic peptides, RPC is generally employed both for the initial analysis and the final large scale purification. HPLC techniques are then introduced at the later stages following initial precipitation, clarification and preliminary separations using soft gel. Purification protocols therefore need to be tailored to the specific target molecule. The key factor that underpins the development of a successful separation protocol is the ability to manipulate the retention of the target molecule so that it can be resolved from other contaminating components.

This chapter thus provides an outline of the general theory of chromatography and the factors that control both the retention time and peakwidth of solutes undergoing separation in terms of the parameters that control resolution. This information can then be used to understand the approaches used to perform separations with specific modes of chromatography as outlined in the remaining chapters in this book

HPLC of Peptides and Proteins

Volume 251

Methods and Protocols

Edited by

Marie-Isabel Aguilar

Article Micropreparative HPLC of peptides and proteins

Because HPLC is a 'comparative' technology you will have to find a pure primary reference standard 1st rather than asking for a protocol. Try the USP or EDQM.

I have previously presented insulin analysis method by HPLC-photometric detection (please see file; Insulin RP-HPLC). It seems interesting that the use of smaller particle-diameter silica gel may be superior to protein analysis method, which uses 10 micro-meter particle-diameter gel (please see file; Lysozyme by HPLC).

You can always use a 99% pure Sigma standard if it is identified and qualified (MS, UV, FTIR, NMR...). These are usually on their 'Certificate of Analysis'.

Bradykinin potentiating peptide has not been sold by Sigma (USA) or Peptide Institude (Japan).

Therefore, you can synthesis it if you have a peptide synthesizer, or ask to Brazil.

LmrBPP9: A synthetic bradykinin-potentiating peptide from Lachesis muta rhombeata venom that inhibits the angiotensin-converting enzyme activity in vitro and reduces the blood pressure of hypertensive rats. Pinheiro-Júnior EL, Boldrini-França J, de Campos Araújo LMP, Santos-Filho NA, Bendhack LM, Cilli EM, Arantes EC. School of Pharmaceutical Sciences of Ribeirão Preto (FCFRP), University of São Paulo, Ribeirão Preto, SP, Brazil. Chemistry Institute, São Paulo State University, Araraquara, SP, Brazil. School of Pharmaceutical Sciences of Ribeirão Preto (FCFRP), University of São Paulo, Ribeirão Preto, SP, Brazil. Electronic address: [email protected]. Bradykinin-potentiating peptides (BPPs) are an important group of toxins present in Lachesis muta rhombeata venom. They act directly at renin-angiotensin-aldosterone system, through the inhibition of angiotensin-converting enzyme (ACE). This action may contribute to the hypotensive shock observed during the envenoming by this species. Thus, the main goal of this study was the solid-phase synthesis of a BPP found in L. m. rhombeata venom and its in vitro and in vivo characterization in relation to ACE inhibition and hypotensive activity, respectively. The LmrBPP9 peptide was synthesized using an automated solid-phase peptide synthesizer and purified by reversed-phase fast protein liquid chromatography (FPLC). The in vitro IC50 of the synthetic peptide is 4.25 ± 0.10 μM, showing a great capacity of ACE inhibition. The in vivo studies showed that LmrBPP9 induces blood pressure reduction, both in normotensive and hypertensive rats, being more pronounced in the last ones. These results agree with the in vitro results, showing that the synthetic peptide LmrBPP9 is a potential molecule to the development of a new antihypertensive drug.

Dear @Samyr Querobino

HPLC is extremely versatile for the isolation of peptides from a wide variety of synthetic or biological sources. The number of applications of HPLC in peptide and protein purification continue to expand at an extremely rapid rate. Solid-phase peptide synthesis and recombinant DNA techniques have allowed the production of large quantities of peptides which need to be highly purified. The design of multidimensional purification schemes to achieve high levels of product purity further highlight the power of HPLC techniques in the analysis and isolation of peptide samples. The complexity of the mixture to be chromatographed depends on the nature of the

source and the degree of preliminary clean-up that can be performed. In the case of synthetic peptides, RPC is generally employed both for the initial analysis and the final large scale purification. HPLC techniques are then introduced at the later stages following initial precipitation, clarification and preliminary separations using soft gel. Purification protocols therefore need to be tailored to the specific target molecule. The key factor that underpins the development of a successful separation protocol is the ability to manipulate the retention of the target molecule so that it can be resolved from other contaminating components.

This chapter thus provides an outline of the general theory of chromatography and the factors that control both the retention time and peakwidth of solutes undergoing separation in terms of the parameters that control resolution. This information can then be used to understand the approaches used to perform separations with specific modes of chromatography as outlined in the remaining chapters in this book

HPLC of Peptides and Proteins

Volume 251

Methods and Protocols

Edited by

Marie-Isabel Aguilar

Article Micropreparative HPLC of peptides and proteins