3 research outputs found
Control of Enzyme–Solid Interactions via Chemical Modification
Electrostatic forces could contribute significantly toward
enzyme–solid
interactions, and controlling these charge–charge interactions
while maintaining high affinity, benign adsorption of enzymes on solids
is a challenge. Here, we demonstrate that chemical modification of
the surface carboxyl groups of enzymes can be used to adjust the net
charge of the enzyme and control binding affinities to solid surfaces.
Negatively charged nanosolid, α-ZrÂ(HPO<sub>4</sub>)<sub>2</sub>·H<sub>2</sub>O (abbreviated as α-ZrP) and two negatively
charged proteins, glucose oxidase (GO) and methemoglobin (Hb), have
been chosen as model systems. A limited number of the aspartate and
glutamate side chains of these proteins are covalently modified with
tetraethylenepentamine (TEPA) to convert these negatively charged
proteins into the corresponding positively charged ones (cationized).
Cationized proteins retained their structure and activities to a significant
extent, and the influence of cationization on binding affinities has
been tested. Cationized GO, for example, showed 250-fold increase
in affinity for the negatively charged α-ZrP, when compared
to that of the unmodified GO, and cationized Hb, similarly, indicated
26-fold increase in affinity. Circular dichroism spectra showed that
α-ZrP-bound cationized GO retained native-like structure to
a significant extent, and activity studies showed that cationized
GO/α-ZrP complex is ∼2.5-fold more active than GO/α-ZrP.
Cationized Hb/α-ZrP retained ∼75% of activity of Hb/α-ZrP.
Therefore, enzyme cationization enhanced affinities by 1–2
orders of magnitude, while retaining considerable activity for the
bound biocatalyst. This benign, chemical control over enzyme charge
provided a powerful new strategy to rationally modulate enzyme–solid
interactions while retaining their biocatalytic properties
Metal-Enzyme Frameworks: Role of Metal Ions in Promoting Enzyme Self-Assembly on α‑Zirconium(IV) Phosphate Nanoplates
Previously, an ion-coupled protein binding (ICPB) model
was proposed
to explain the thermodynamics of protein binding to negatively charged
α-ZrÂ(IV) phosphate (α-ZrP). This model is tested here
using glucose oxidase (GO) and met-hemoglobin (Hb) and several cations
(ZrÂ(IV), CrÂ(III), AuÂ(III), AlÂ(III), CaÂ(II), MgÂ(II), ZnÂ(II), NiÂ(II),
NaÂ(I), and HÂ(I)). The binding constant of GO with α-ZrP was
increased ∼380-fold by the addition of either 1 mM ZrÂ(IV) or
1 mM CaÂ(II), and affinities followed the trend ZrÂ(IV) ≃ CaÂ(II)
> CrÂ(III) > MgÂ(II) ≫ HÂ(I) > NaÂ(I). Binding studies
could not
be conducted with AuÂ(III), AlÂ(III), ZnÂ(II), CuÂ(II), and NiÂ(II), as
these precipitated both proteins. ZrÂ(IV) increased Hb binding constant
to α-ZrP by 43-fold, and affinity enhancements followed the
trend ZrÂ(IV) > HÂ(I) > MgÂ(II) > NaÂ(I) > CaÂ(II) > CrÂ(III).
Zeta potential
studies clearly showed metal ion binding to α-ZrP and affinities
followed the trend, ZrÂ(IV) ≫ CrÂ(III) > ZnÂ(II) > NiÂ(II)
> MgÂ(II)
> CaÂ(II) > AuÂ(III) > NaÂ(I) > HÂ(I). Electron microscopy
showed highly
ordered structures of protein/metal/α-ZrP intercalates on micrometer
length scales, and protein intercalation was also confirmed by powder
X-ray diffraction. Specific activities of GO/ZrÂ(IV)/α-ZrP and
Hb/ZrÂ(IV)/α-ZrP ternary complexes were 2.0 × 10<sup>–3</sup> and 6.5 × 10<sup>–4</sup> M<sup>–1</sup> s<sup>–1</sup>, respectively. While activities of all GO/cation/α-ZrP
samples were comparable, those of Hb/cation/α-ZrP followed the
trend MgÂ(II) > NaÂ(I) > HÂ(I) > CrÂ(III) > CaÂ(II) ≃
ZrÂ(IV). Metal
ions enhanced protein binding by orders of magnitude, as predicted
by the ICPB model, and binding enhancements depended on charge as
well as the phosphophilicity/oxophilicity of the cation
Multicolor Monitoring of the Proteasome’s Catalytic Signature
The proteasome, a validated anticancer
target, participates in
an array of biochemical activities, which range from the proteolysis
of defective proteins to antigen presentation. We report the preparation
of biochemically and photophysically distinct green, red, and far-red
real-time sensors designed to simultaneously monitor the proteasome’s
chymotrypsin-, trypsin-, and caspase-like activities, respectively.
These sensors were employed to assess the effect of simultaneous multiple
active site catalysis on the kinetic properties of the individual
subunits. Furthermore, we have found that the catalytic signature
of the proteasome varies depending on the source, cell type, and disease
state. Trypsin-like activity is more pronounced in yeast than in mammals,
whereas chymotrypsin-like activity is the only activity detectable
in B-cells (unlike other mammalian cells). Furthermore, chymotrypsin-like
activity is more prominent in transformed B cells relative to their
counterparts from healthy donors