Bactenecin 7 peptide fragment as a tool for intracellular delivery of a phosphorescent oxygen sensor.
Research on cell-penetrating peptides for the intracellular delivery of porphyrin compounds has mainly focused on the use of trans-activator of transcription (TAT)-derived peptides and, to a lesser extent, on proline-rich peptides and phosphorescent metalloporphyrins. In this article, we describe a novel phosphorescent oxygen-sensitive probe for intracellular use which comprises a bactenecin 7 peptide fragment (15-24) conjugated with the uncharged monofunctional derivative of Pt(II) coproporphyrin I (PEPP0). This probe provides efficient loading of various mammalian cells, including PC12, HCT116, SH-SY5Y and HeLa, via cell-type-dependent uptake mechanisms. The conjugate displays a similar distribution in cytoplasm and mitochondria which allows local oxygen levels to be monitored. Respiratory responses of PC12 cells loaded with the conjugate, measured on a time-resolved fluorescent reader, showed significant cell deoxygenation in response to uncoupling by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone and external hypoxia. Treatment with mitochondrial inhibitors led to a decrease in cell deoxygenation. Although the biophysical properties of this conjugate are similar to those of the phosphorescent intracellular oxygen-sensitive probes described previously, it possesses a number of advantages, including ease of synthesis, high loading efficiency and reliability in physiological experiments with cells. This intracellular probe can be employed for the measurement of intracellular O(2) levels in samples containing mammalian cells using the phosphorescence quenching technique. In addition, the responses to metabolic stimuli can be assessed in a wide range of cells, as can the levels of relative cell oxygenation under external hypoxia.
Dmitriev
Ruslan I
RI
Biochemistry Department, University College Cork, Cork, Ireland. r.dmitriev@ucc.ie
Ropiak
Honorata M
HM
Yashunsky
Dmitri V
DV
Ponomarev
Gelii V
GV
Zhdanov
Alexander V
AV
Papkovsky
Dmitri B
DB
D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry / Q000378:metabolism*; D015374:Biosensing Techniques; D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015394:Molecular Structure; D010100:Oxygen / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats
D000890:Anti-Infective Agents; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D020033:Protein Isoforms; C056454:bactenecin; D010100:Oxygen