Mechanistic investigation of N-homocysteinylation-mediated protein-gold nanoconjugate assembly.
Herein we report the use of protein-gold nanoconjugate (PGNs) as probes for elucidating mechanistic events involved in protein homocystamide detection with gold nanoparticles (GNPs), as was previously reported by our laboratory. Three different PGN probes are synthesized by direct adsorption of cytochrome c, albumin, or human serum onto citrate-capped GNPs. The PGNs are subsequently purified and treated to confer N-homocysteinylation. Individual PGN systems are evaluated to assess the effect of modification on (1) surface plasmon resonance (SPR), (2) protein structural conformation, and (3) assembly-association. The degree of PGN assembly and colorimetric signal observed postmodification varies based on the type of conjugated protein. For example, results of time-resolved dynamic light scattering studies indicate that modification of cytochrome c-PGNs yields rapid formation of macroscopic nanoparticle assemblies that eventually precipitate from solution. In contrast, albumin and human serum PGNs exhibit higher stability toward modification. Additionally, findings from circular dichroism studies indicate significant modification-induced denaturation, which is what may initiate assembly via electrosteric destabilization of PGNs. The results of electrophoretic studies appear to confirm that the process of N-homocysteinylation-mediated PGN assembly culminates in covalent interparticle association by disulfide cross-linking among modified proteins.
Gates
Arther T
AT
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
Moore
Leonard
L
Sylvain
Monica R
MR
Jones
Christina M
CM
Lowry
Mark
M
El-Zahab
Bilal
B
Robinson
James W
JW
Strongin
Robert M
RM
Warner
Isiah M
IM
D004396:Coloring Agents / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D006710:Homocysteine / Q000737:chemistry*; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry*; D015394:Molecular Structure; D012709:Serum Albumin / Q000737:chemistry
D004396:Coloring Agents; D012709:Serum Albumin; D006710:Homocysteine; D006046:Gold