Tuning Enzyme/α-Zr(IV) Phosphate Nanoplate Interactions via Chemical Modification of Glucose Oxidase.

Using glucose oxidase (GOx) and α-Zr(IV) phosphate nanoplates (α-ZrP) as a model system, a generally applicable approach to control enzyme-solid interactions via chemical modification of amino acid side chains of the enzyme is demonstrated. Net charge on GOx was systematically tuned by appending different amounts of polyamine to the protein surface to produce chemically modified GOx(n), where n is the net charge on the enzyme after the modification and ranged from -62 to +95 electrostatic units in the system. The binding of GOx(n) with α-ZrP nanosheets was studied by isothermal titration calorimetry (ITC) as well as by surface plasmon resonance (SPR) spectroscopy. Pristine GOx showed no affinity for the α-ZrP nanosheets, but GOx(n) where n ≥ -20 showed binding affinities exceeding (2.1 ± 0.6) × 106 M-1, resulting from the charge modification of the enzyme. A plot of GOx(n) charge vs Gibbs free energy of binding (ΔG) for n = +20 to n = +65 indicated an overall increase in favorable interaction between GOx(n) and α-ZrP nanosheets. However, ΔG is less dependent on the net charge for n > +45, as evidenced by the decrease in the slope as charge increased further. All modified enzyme samples and enzyme/α-ZrP complexes retained a significant amount of folding structure (examined by circular dichroism) as well as enzymatic activities. Thus, strong control over enzyme-nanosheet interactions via modulating the net charge of enzymes may find potential applications in biosensing and biocatalysis.

Correlation of Ecom50 values between mass spectrometers: effect of collision cell radiofrequency voltage on calculated survival yield.

RATIONALE: The determination of the center-of-mass energy at which 50% of a precursor ion decomposes (Ecom(50)) during collision-induced dissociation (CID) is dependent on the chemical structure of the ion as well as the physical and electrical characteristics of the collision cell. The current study was designed to identify variables influencing Ecom(50) values measured on four different mass spectrometers. METHODS: Fifteen test compounds were protonated using + ve electrospray ionization and the resulting ions were fragmented across a range of collision energies by CID. Survival yield versus collision energy curves were then used to calculate Ecom(50) values for each of these [M+H](+) ions on four different mass spectrometers. In addition, the relative recovery of the [M+H](+) ions of eight compounds ranging in molecular weight from 46 to 854 Da were determined at collision cell radiofrequency (RF) voltages ranging from 0 to 600 V. RESULTS: Ecom(50) values determined on the four instruments were highly correlated (r(2) values ranged from 0.953 to 0.992). Although these overall correlations were high, we found different maximum ion recoveries depending on collision cell RF voltage. High-mass ions had greater recovery at higher collision cell RF voltages, whereas low-mass ions had greater recovery at lower collision cell RF voltages as well as a broader range of ion recoveries. CONCLUSIONS: Ecom(50) values measured on four different instruments correlated surprisingly well given the differences in electrical and physical characteristics of the collision cells. However, our results suggest caution when comparing Ecom(50) values or CID spectra between instruments without correcting for the effects of RF voltage on ion transfer efficiency.

One-step preparation of bioactive enzyme/inorganic materials.

Simultaneous exfoliation of crystalline α-zirconium phosphate (α-ZrP) nanosheets and enzyme binding, induced by shearing, without the addition of any toxic additives is reported here for the first time. These materials were thoroughly characterized and used for applications. The bulk α-ZrP material (20 mg mL-1) was exfoliated with low concentrations of a protein such as bovine serum albumin (BSA, 3 mg mL-1) in a shear reactor at 10k rpm for <80 minutes. Exfoliation was monitored by powder X-ray diffraction with samples displaying a gradual but complete loss of the 7.6 Å (002) peak, which is characteristic of bulk α-ZrP. The fully exfoliated sample loaded with the protein was characterized by transmission and scanning electron microscopy in addition to other biophysical methods. Lysozyme, glucose oxidase, met-hemoglobin, and ovalbumin also induced exfoliation and directly produced enzyme/ZrP biocatalysts. Thus, exfoliation, biophilization and enzyme binding are accomplished in a single step. Several factors contributed to the exfoliation kinetics, and the rate increased with α-ZrP and BSA concentrations and decreased with pH. However, the exfoliation efficiency inversely depended on the isoelectric point of the protein with ovalbumin (pI = 4.5) being the best and lysozyme (pI = 11.1) being the worst. A strong correlation between the protein size and exfoliation efficiency was noted, and the latter suggests the role of hydrodynamic factors in the process. Exfoliation was also achieved by simple stirring using a magnetic stirrer, under low volumes, and model enzymes, indicating 60-90% retention of bound enzymatic activities. The addition of BSA to enzymes as the diluent and stabilizing agent also prevents enzymes from the denaturing effect caused by stirring. This new method requires no pre-treatment of α-ZrP with toxic exfoliating agents such as tetrabutyl ammonium hydroxide and provides bioactive enzyme/inorganic materials in a single step. These protein-loaded biocompatible nanosheets may be useful for biocatalysis and biomedical applications.

Designer Histone Complexes: Controlling Protein-DNA Interactions with Protein Charge as an "All-or-None" Digital Switch.

An artificial histone is synthesized that functions as a DNA-protein digital switch, where DNA binding is all or none, controlled by a sharp threshold of protein charge. A non-DNA-binding protein, glucose oxidase (GOx), was chemically modified by attaching an increasing number of triethylenetetramine (TETA) side chains to its glutamate/aspartate groups to obtain a small library of covalently modified GOx(n) derivatives. The parameter n denotes the net charge on the protein at pH 7, which was increased from -62 (pristine GOx) to +75 by attaching an increasing number of TETA residues to the protein. All GOx(n) derivatives retained their secondary structure to a good extent, as monitored by UV circular dichroism (CD) spectroscopy, and they also retained oxidase activities to a significant extent. The interaction of the GOx(n) with calf thymus DNA was examined by isothermal titration calorimetry (ITC). Pristine GOx of -62 charge at pH 7 in 10 mM Tris-HCl and 50 mM NaCl buffer had no affinity for the negatively charged DNA helix, and GOx(n) with n < +30 had no affinity for DNA either. However, binding has been turned on abruptly when n ≥ +30 with binding constants (Kb) ranging from (1.5 ± 0.7) × 107 to (7.3 ± 2.8) × 107 M-1 for n values of +30 and +75, respectively, and this type of "all-or-none" binding based on protein charge is intriguing. Furthermore, thermodynamic analysis of the titration data revealed that binding is entirely entropy-driven with ΔS ranging from 0.09 ± 0.007 to 0.19 ± 0.008 kcal/mol K with enthalpic penalties of 17.0 ± 2.3 and 46.1 ± 2.1 kcal/mol, respectively. The binding had intrinsic propensities (ΔG) ranging from -9.8 ± 0.14 to -10.7 ± 0.25 kcal/mol, independent of n. DNA binding distorted protein-DNA secondary structure, as evidenced by CD spectroscopy, but oxidase activity of GOx(n)/DNA complexes has been unaffected. This is the very first example of an artificial histone (GOx(n)) where the protein charge functioned as a DNA-binding switch; protein charge is in turn under complete chemical control while preserving the biological activity of the protein. The new insight gained here could be useful in the design of novel "on-off" protein switches.