Gold nanoparticle-conjugated pepsin for efficient solution-like heterogeneous biocatalysis in analytical sample preparation protocols.
Immobilization of enzymes on mesoporous microparticulate carriers has traditionally been accompanied by reduction in enzyme activity. Herein, we document that immobilization of pepsin via amide coupling on gold nanoparticles (GNPs) with a carboxy-terminated hydrophilic PEG7 shell resulted in a heterogeneous nanobiocatalyst with essentially equivalent turnover rates, k cat (90 %), and enhanced catalytic efficiencies, k cat/K M (107 %), compared to homogeneous catalysis with pepsin in free solution for cytochrome C as model substrate. This heterogeneous catalyst showed further at least equivalent bioactivity in a digestion reaction of a protein mixture consisting of cytochrome C, bovine serum albumin, and myoglobin. UHPLC-ESI-QTOF-MS/MS analysis of the digests with subsequent Mascot database search allowed unequivocal identification of all proteins with high score and good sequence coverage. The functionalized nanoparticles were further characterized by Vis spectroscopy in terms of the surface plasmon resonance (SPR) band, by dynamic light scattering (DLS) with regard to hydrodynamic diameters, and in view of their ζ potentials at each step of synthesis and surface modification. These measurements also revealed that the pepsin-functionalized GNPs were sufficiently stable over at least 1 month; thus providing a satisfactory shelf life to the heterogeneous catalyst. Advantageously, the pepsin-GNP bioconjugate can be conveniently removed after reaction by simple centrifugation steps which makes them a useful tool for analysis of therapeutic peptides and proteins, including monoclonal antibodies. The practical utility of the nanobiocatalyst was documented by digestion of a monoclonal antibody which yielded the F(ab')2 fragment with a mass of 97,619.4 Da. Graphical Abstract Pepsin conjugated to pegylated gold nanoparticles exhibit properties of homogeneous catalysis such as enzyme activity like pepsin in solution and ability to pipette the stable colloidal suspension whereas one can take benefit from easy removal by centrifugation and re-use which are characteristics of heterogeneous catalysis.
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Markus
M
Höldrich
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Adrian
A
Sievers-Engler
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany. Michael.laemmerhofer@uni-tuebingen.de.
Michael
M
Lämmerhofer
D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006046:Gold / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry*; D058726:Nanoconjugates / Q000737:chemistry; D010434:Pepsin A / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry; D013048:Specimen Handling / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000911:Antibodies, Monoclonal; D058726:Nanoconjugates; D012996:Solutions; D006046:Gold; D010434:Pepsin A
Bioconjugate; Enzyme kinetics; Gold nanoparticle; Heterogeneous nanobiocatalyst; Monoclonal antibody fragment F(ab')2; Quadrupole time-of-flight mass spectrometry