cytochrome-c-t and betadex

The ability to design surfaces with reversible, high-affinity protein binding sites represents a significant step forward in the advancement of analytical methods for diverse biochemical and biomedical applications. Herein, we report a dynamic supramolecular strategy to directly assemble proteins on surfaces based on multivalent host-guest interactions. The host-guest interactions are achieved by one-step nanofabrication of a well-oriented β-cyclodextrin host-derived self-assembled monolayer on gold (β-CD-SAM) that forms specific inclusion complexes with hydrophobic amino acids located on the surface of the protein. Cytochrome c, insulin, α-chymotrypsin, and RNase A are used as model guest proteins. Surface plasmon resonance and static time-of-flight secondary ion mass spectrometry studies demonstrate that all four proteins interact with the β-CD-SAM in a specific manner via the hydrophobic amino acids on the surface of the protein. The β-CD-SAMs bind the proteins with high nanomolar to single-digit micromolar dissociation constants ( K

Topics: beta-Cyclodextrins; Cytochromes c; Gold; Immobilized Proteins; Insulin; Protein Binding; Ribonuclease, Pancreatic; Spectrometry, Mass, Secondary Ion; Surface Plasmon Resonance; Surface Properties

We describe a novel method for rapid and ultrasensitive detection of intact glycoproteins without enzymatic pretreatment which was commonly used in proteomic research. This method is based on using gold nanoparticle (AuNP) as signal tag in laser desorption/ionization mass spectrometry (LDI-MS) analysis combined with boronic acid assisted isolation strategy. Briefly speaking, target glycoproteins were firstly isolated from sample solution with boronic acid functionalized magnetic microparticles, and then the surface modified gold nanoparticles were added to covalently bind to the glycoproteins. After that, these AuNP tagged glycoproteins were eluted from magnetic microparticles and applied to LDI-MS analysis. The mass signal of AuNP rather than that of glycoprotein was detected and recorded in this strategy. Through data processing of different standard glycoproteins, we have demonstrated that the signal of AuNP could be used to quantitatively represent glycoprotein. This method allows femtomolar detection of intact glycoproteins. We believe that the successful validation of this method on three different kinds of glycoproteins suggests the potential use for tracking trace amount of target glycoproteins in real biological samples in the near future.

Topics: beta-Cyclodextrins; Boronic Acids; Caseins; Cytochromes c; Glycoproteins; Gold; Limit of Detection; Metal Nanoparticles; Myoglobin; Reproducibility of Results; Sensitivity and Specificity; Serum Albumin, Bovine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Topics: Animals; beta-Cyclodextrins; Cytochromes c; Horses; Models, Molecular; Protein Binding

Cyclodextrins (CDs) are cyclic oligosaccharides that are able to form inclusion complexes with a variety of substances. For pharmaceutical applications, CD-based drug formulations offer important advantages compared with uncomplexed drugs. These include improved water solubility of lipophilic drug molecules, increased chemical stability, as well as enhanced bioavailability and absorption rate. Also, a number of topical formulations for dermal and transdermal drug delivery contain CDs. However, the most frequently used CDs - beta-CD and MbetaCD - are known to extract cholesterol from plasma membranes and thus to cause cellular damage and cell death. In the present study, the influence of various CDs and CD derivatives on the human keratinocyte cell line HaCaT was assessed. We found that beta-CD and MbetaCD induce apoptosis via the activator caspase-8, which subsequently activates the effector caspases-3/-7. Furthermore, beta-CD-induced apoptosis is accompanied by mitochondrial cytochrome c release. A significant shift from mitochondria into the cytosol was found. These findings may provide further rationale to the use of CDs in topical formulations for dermal and transdermal drug delivery or as raw material in order to functionalize textiles for medical applications.

Topics: Administration, Cutaneous; Apoptosis; beta-Cyclodextrins; Caspase 8; Cell Line; Cytochromes c; Drug Delivery Systems; Flow Cytometry; Humans; Keratinocytes; Mitochondria