The Protein Journal

, Volume 32, Issue 6, pp 456–466 | Cite as

Improved Protease Stability of the Antimicrobial Peptide Pin2 Substituted with d-Amino Acids

  • G. Carmona
  • A. Rodriguez
  • D. Juarez
  • G. Corzo
  • E. Villegas


Cationic antimicrobial peptides (AMPs) have attracted a great interest as novel class of antibiotics that might help in the treatment of infectious diseases caused by pathogenic bacteria. However, some AMPs with high antimicrobial activities are also highly hemolytic and subject to proteolytic degradation from human and bacterial proteases that limit their pharmaceutical uses. In this work a d-diastereomer of Pandinin 2, d-Pin2, was constructed to observe if it maintained antimicrobial activity in the same range as the parental one, but with the purpose of reducing its hemolytic activity to human erythrocytes and improving its ability to resist proteolytic cleavage. Although, the hydrophobic and secondary structure characteristics of l- and d-Pin2 were to some extent similar, an important reduction in d-Pin2 hemolytic activity (30–40 %) was achieved compared to that of l-Pin2 over human erythrocytes. Furthermore, d-Pin2 had an antimicrobial activity with a MIC value of 12.5 μM towards Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and two strains of Pseudomonas aeruginosa in agar diffusion assays, but it was half less potent than that of l-Pin2. Nevertheless, the antimicrobial activity of d-Pin2 was equally effective as that of l-Pin2 in microdilution assays. Yet, when d- and l-Pin2 were incubated with trypsin, elastase and whole human serum, only d-Pin2 kept its antimicrobial activity towards all bacteria, but in diluted human serum, l- and d-Pin2 maintained similar peptide stability. Finally, when l- and d-Pin2 were incubated with proteases from P. aeruginosa DFU3 culture, a clinical isolated strain, d-Pin2 kept its antibiotic activity while l-Pin2 was not effective.


d-diastereomer Peptide Antimicrobial Hemolysis Enzyme degradation 



Reverse-phase HPLC


High pressure liquid chromatography


Mueller–Hinton broth


Mueller–Hinton agar


Colony forming units


Circular dichroism


Cationic antimicrobial peptide


Minimal inhibitory concentrations


Trifluoroacetic acid


Phosphate buffer saline, pH 7.2


Polyacrylamide gel electrophoresis


Sodium dodecyl sulfate





We acknowledge Dr. Fernando Zamudio for mass spectrometric determinations and QBP Ma. Rocio Patiño Maya for CD spectra data acquisition. Gabriela Carmona was recipient of an MSc scholarship 30175 from CONACyT (226330). Daniel Juaréz was recipient of an undergraduate scholarship from RedFarmed-CONACyT. Alexis Rodríguez was recipient of a PhD scholarship 24492 from CONACyT (172816), and now he is supported by a postdoctoral fellowship from PROMEP. This work was also supported by CONACyT proyect Grants 153606 to GC, and 83962/2007 and 106949/2008 to Elba Villegas.

Supplementary material

10930_2013_9505_MOESM1_ESM.tif (5 mb)
Mass spectra of L-Pin2 (A) D-Pin2 (B). (TIFF 5082 kb)
10930_2013_9505_MOESM2_ESM.tif (545 kb)
Zymogram of proteases from P. aeruginosa strains ATCC 27853 and DFU3. On the right side, the first four lanes of the gel correspond to the proteins from the supernatant from a culture of P. aeruginosa strains ATCC 27853. The following four lanes in the same gel correspond to the proteins from the supernatant from a culture of P. aeruginosa strains DFU3. On the left side, the molecular weight markers are shown. (TIFF 544 kb)
10930_2013_9505_MOESM3_ESM.tif (3.1 mb)
Twenty-four N-terminal degradation cycles of D-Pin2. Reverse-phase HPLC chromatograms of each phenylthiohydantoin (PTH)-amino acid. The magenta profile represents the separation of the PTH-amino acid from the N-terminal degradation of D-Pin2, and the black profile represents PTH-L-amino acid standards. Some HPLC chromatograms were zoomed into observe in detail the eluting peaks. The cycle 01 contains also a peak that elutes within the Gln elution time as a consequence of the polybrene reaction. DMPTU, the peak at 8.5 min and DPTU are secondary products of the N-terminal reaction. (TIFF 3128 kb)
10930_2013_9505_MOESM4_ESM.tif (3.1 mb)
Stability of D-Pin2 in the presence of trypsin and elastase. After enzyme treatment (4 h) with either trypsin (A) and elastase (B) (see Materials and methods), D-Pin2 was separated using an analytical reverse phase C18 column, at a flow rate of 1 mL/min, with linear gradients from 15-60 % B in 45 min (TIFF 3128 kb)
10930_2013_9505_MOESM5_ESM.tif (104 kb)
Stability of D-Pin2 in the presence of Pseudomonas aeruginosa DFU3 proteases. D-Pin2 was incubated in the presence of P. aeruginosa DFU3 proteases. Samples were taken at 0, 4, 8, 12 and 24 h (A, B, C, D and E, respectively) and separated using an analytical reverse phase C18 column, at a flow rate of 1 mL/min, with linear gradients from 15-60 % B in 45 min (absorbance at A230). (TIFF 104 kb)


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • G. Carmona
    • 1
  • A. Rodriguez
    • 1
  • D. Juarez
    • 1
  • G. Corzo
    • 2
  • E. Villegas
    • 1
  1. 1.Laboratorio de Estructura-Funcion e Ingenieria de ProteinasCentro de Investigacion en Biotecnologia Universidad Autonoma del Estado de MorelosCuernavacaMexico
  2. 2.Departamento de Medicina Molecular y BioprocesosInstituto de Biotecnologia, UNAMCuernavacaMexico

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