Protamine as an efficient membrane-translocating peptide.
Protamine, a mixture of positively charged proteins from salmon roe used in diverse pharmaceutical applications, was reacted with the N-hydroxysuccinimide ester of tetramethylrhodamine to yield tetramethylrhodamine-labeled protamines (Pro(Rh)) containing one mole of fluorochrome per mole of protein. The internalization of tetramethylrhodamine-labeled protamine (Pro(Rh)) and the fluorescein-labeled tat peptide (Tat(Fl)) showed a similar dependence on time and concentration. Pro(Rh) and Tat(Fl) showed strong nuclear localizations, evident with both live cells and fixed cells co-stained with DAPI, a nuclear stain. The loss of fluorescence when cells were loaded with Pro(Rh) or Tat(Fl) was similar, further supporting a strong similarity between these two materials. Finally, when Pro(Ph) was covalently attached to the amino-CLIO nanoparticle, the cellular uptake of the nanoparticle was greatly enhanced. All experiments were performed with HeLa and CaCo-2 cells with similar results. These observations imply that protamine, a protein in regular clinical use, might be used for the design of novel membrane translocating/nuclear localizing pharmaceuticals whose development was initiated with other membrane-translocating peptides. In addition, the fluorescent protamines developed here might be used to further our understanding of this important pharmaceutical.
Reynolds
Fred
F
Center for Molecular Imaging Research, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02129, USA.
Weissleder
Ralph
R
Josephson
Lee
L
D000818:Animals; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism*; D029941:Fish Proteins / Q000737:chemistry / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D015696:Gene Products, tat / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011479:Protamines / Q000737:chemistry* / Q000378:metabolism*; D021381:Protein Transport; D012235:Rhodamines / Q000737:chemistry; D012474:Salmon
D029941:Fish Proteins; D015696:Gene Products, tat; D010446:Peptide Fragments; D011479:Protamines; D012235:Rhodamines; D019793:Fluorescein