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Development and application of a label-free fluorescence method for determining the composition of gold nanoparticle-protein conjugates.

Sotnikov DV, Zherdev AV, Dzantiev BB - Int J Mol Sci (2014)

Bottom Line: The equilibrium dissociation constants of the gold nanoparticle conjugates with IgG, BSA, protein G, STI in the initial section of the concentration dependence curve were 4, 6, 10, and 15 nM, respectively.Close to saturation, the corresponding values were 25, 76, 175, and 100 nM, respectively.The maximal binding capacities of a single gold nanoparticle for IgG, BSA, Protein G, and STI were 52, 90, 500, and 550, respectively, which agrees well with the hypothesis of monolayer immobilization.

View Article: PubMed Central - PubMed

Affiliation: A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, Moscow 119071, Russia. sotnikov-d-i@mail.ru.

ABSTRACT
A method was developed for determining the composition of the conjugates between gold nanoparticles and proteins based on the intrinsic fluorescence of unbound protein molecules. The fluorescence was evaluated after separation of the conjugates from the reaction mixture by centrifugation. Gold nanoparticles obtained using the citrate technique (average diameter 24 nm) were conjugated at pH 5.4 with the following four proteins: human immunoglobulin G (IgG), bovine serum albumin (BSA), recombinant streptococcal protein G (protein G), and Kunitz-type soybean trypsin inhibitor (STI). The compositions of these conjugates were determined using the developed method. The conjugate compositions were dependent on the concentration of the added protein, and in all cases reached saturation. The equilibrium dissociation constants of the gold nanoparticle conjugates with IgG, BSA, protein G, STI in the initial section of the concentration dependence curve were 4, 6, 10, and 15 nM, respectively. Close to saturation, the corresponding values were 25, 76, 175, and 100 nM, respectively. The maximal binding capacities of a single gold nanoparticle for IgG, BSA, Protein G, and STI were 52, 90, 500, and 550, respectively, which agrees well with the hypothesis of monolayer immobilization.

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Images of the proteins used in the study and their conjugates with gold nanoparticles (ø 24 nm). (A,B) for immunoglobulin G (IgG); (C,D) for bovine serum albumin (BSA); (E,F) for Kunitz-type soybean trypsin inhibitor (STI); (G,H) for recombinant streptococcal protein G (protein G). Figures B, D, F, H show orientations of proteins on the nanoparticle surface for models given at figures A, C, E, G, respectively.
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ijms-16-00907-f007: Images of the proteins used in the study and their conjugates with gold nanoparticles (ø 24 nm). (A,B) for immunoglobulin G (IgG); (C,D) for bovine serum albumin (BSA); (E,F) for Kunitz-type soybean trypsin inhibitor (STI); (G,H) for recombinant streptococcal protein G (protein G). Figures B, D, F, H show orientations of proteins on the nanoparticle surface for models given at figures A, C, E, G, respectively.

Mentions: As an illustration of this hypothesis, we constructed three-dimensional models of these conjugates. A sphere 24 nm in diameter was created as an analog of the nanoparticle; protein models were placed on its surface. Figure 7 shows models of the colloid conjugates with coverage of 1/8th of the nanoparticle surface. The following numbers of protein molecules were placed on the nanoparticle surface: IgG-6; BSA-11; STI-60; and protein G-63. Thus, for the given density of surface coverage, the total number of protein molecules for the entire sphere was 48, 88, 480 and 504 molecules for IgG, BSA, STI, and protein G, respectively. These values are comparable to the data in Table 1. Thus, the experimentally determined amounts of adsorbed proteins for the saturation conditions could form a protein monolayer on the surface of the studied nanoparticle.


Development and application of a label-free fluorescence method for determining the composition of gold nanoparticle-protein conjugates.

Sotnikov DV, Zherdev AV, Dzantiev BB - Int J Mol Sci (2014)

Images of the proteins used in the study and their conjugates with gold nanoparticles (ø 24 nm). (A,B) for immunoglobulin G (IgG); (C,D) for bovine serum albumin (BSA); (E,F) for Kunitz-type soybean trypsin inhibitor (STI); (G,H) for recombinant streptococcal protein G (protein G). Figures B, D, F, H show orientations of proteins on the nanoparticle surface for models given at figures A, C, E, G, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307282&req=5

ijms-16-00907-f007: Images of the proteins used in the study and their conjugates with gold nanoparticles (ø 24 nm). (A,B) for immunoglobulin G (IgG); (C,D) for bovine serum albumin (BSA); (E,F) for Kunitz-type soybean trypsin inhibitor (STI); (G,H) for recombinant streptococcal protein G (protein G). Figures B, D, F, H show orientations of proteins on the nanoparticle surface for models given at figures A, C, E, G, respectively.
Mentions: As an illustration of this hypothesis, we constructed three-dimensional models of these conjugates. A sphere 24 nm in diameter was created as an analog of the nanoparticle; protein models were placed on its surface. Figure 7 shows models of the colloid conjugates with coverage of 1/8th of the nanoparticle surface. The following numbers of protein molecules were placed on the nanoparticle surface: IgG-6; BSA-11; STI-60; and protein G-63. Thus, for the given density of surface coverage, the total number of protein molecules for the entire sphere was 48, 88, 480 and 504 molecules for IgG, BSA, STI, and protein G, respectively. These values are comparable to the data in Table 1. Thus, the experimentally determined amounts of adsorbed proteins for the saturation conditions could form a protein monolayer on the surface of the studied nanoparticle.

Bottom Line: The equilibrium dissociation constants of the gold nanoparticle conjugates with IgG, BSA, protein G, STI in the initial section of the concentration dependence curve were 4, 6, 10, and 15 nM, respectively.Close to saturation, the corresponding values were 25, 76, 175, and 100 nM, respectively.The maximal binding capacities of a single gold nanoparticle for IgG, BSA, Protein G, and STI were 52, 90, 500, and 550, respectively, which agrees well with the hypothesis of monolayer immobilization.

View Article: PubMed Central - PubMed

Affiliation: A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Prospect 33, Moscow 119071, Russia. sotnikov-d-i@mail.ru.

ABSTRACT
A method was developed for determining the composition of the conjugates between gold nanoparticles and proteins based on the intrinsic fluorescence of unbound protein molecules. The fluorescence was evaluated after separation of the conjugates from the reaction mixture by centrifugation. Gold nanoparticles obtained using the citrate technique (average diameter 24 nm) were conjugated at pH 5.4 with the following four proteins: human immunoglobulin G (IgG), bovine serum albumin (BSA), recombinant streptococcal protein G (protein G), and Kunitz-type soybean trypsin inhibitor (STI). The compositions of these conjugates were determined using the developed method. The conjugate compositions were dependent on the concentration of the added protein, and in all cases reached saturation. The equilibrium dissociation constants of the gold nanoparticle conjugates with IgG, BSA, protein G, STI in the initial section of the concentration dependence curve were 4, 6, 10, and 15 nM, respectively. Close to saturation, the corresponding values were 25, 76, 175, and 100 nM, respectively. The maximal binding capacities of a single gold nanoparticle for IgG, BSA, Protein G, and STI were 52, 90, 500, and 550, respectively, which agrees well with the hypothesis of monolayer immobilization.

Show MeSH
Related in: MedlinePlus