Effects of nanoparticles on the
enzyme activity
Prepared by
Bushra Habeeb Ahmad
1
Nanopartcles
Nanotechnology is defined as the understanding and control of matter
at dimensions of roughly 1to100 nm, where unique physical properties make
novel applications possible. NP are therefore considered substances that are
less than 100 nm in size in more than one dimension. They can be spherical,
tubular, or irregularly shaped and can exist in fused, aggregated or
agglomerated forms. (Figure 1) shows how the NP fit into other size-dependent
categories that have been used for many decades. The commonly used
definition of ‘‘dissolved’’ is in most cases operationally defined by all
compounds passing through a filter, in many cases with a cutoff at 0.45 mm.
The colloidal fraction is defined as having a size between 1 nm and 1 mm,
therefore overlapping with the NP. Separation between dissolved, colloidal and
particulate matter in natural waters is normally given by the availability of
analytical methods that can distinguish among these fractions without
introducing artifacts during the measurement process (1).
Fig. 1. Definitions of different size classes for nanoparticles.
2
Classification of nanoparticles
NP can be divided into natural and anthropogenic particles (Table 1)(2).
Table 1. Classification of nanoparticles
Natura Natural
C-containing
Formation
Biogenic
Geogenic
Atmospheric
Pyrogenic
Inorganic
Biogenic
Geogenic
Anthropogenic C-containing
(manufactured,
engineered)
Atmospheric
By-product
Engineered
Inorganic
By-product
Engineered
Organic colloids
Organisms
Soot
Aerosols
Soot
Oxides
Metals
Oxides
Clays
Aerosols
Combustion
by-products
Examples
Humic, fulvic acids
Viruses
Fullerenes
Organic acids
CNT
Fullerenes
Nanoglobules,onion-shaped nanospheres
Magnetite
Ag, Au
Fe-oxides
Allophane
Sea salt
CNT
Nanoglobules, onion-shaped nanospheres
Soot
Carbon Black
Fullerenes
Functionalized CNT, fullerenes
Polymeric NP
Polyethyleneglycol (PEG) NP
Combustion by-products Platinum group metals
Oxides
TiO2, SiO2
Metals
Ag, iron
Salts
Metal-phosphates
Aluminosilicates
Zeolites, clays, ceramics
Nanoparticle’s structure:
Nanoparticles are composed of two parts: the core material, and a surface
modifier that may be employed to change the physicochemical properties of this
core material. The core materials may be biological materials like peptides,
phospholipids, lipids, lactic acid, dextran or chitosan, or may be formed of a
chemical polymer, carbon, silica, or metals. Nanoparticles have significant
adsorption capacities due to their relatively large surface area, therefore they are
able to bind or carry other molecules such as chemical compounds, drugs,
probes and proteins attached to the surface by covalent bonds or by adsorption.
Hence, the physicochemical properties of nanoparticles, such as charge and
hydrophobicity, can be altered by attaching specific chemical compounds,
peptides or proteins to the surface. The functionality of nanoparticles is thus
enhanced or changed. The efficacy of nanoparticles for any application
dependson the physicochemical characteristics of both their core material and
surface modifiers. The core composition of the nanoparticles, together with
their surface properties, also determines their biocompatibility and their ability
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to be biodegraded (2). The rapid growth in nanotechnology has spurred
significant interest in the environmental applications of nanomaterials.
Nanomaterials are excellent adsorbents, catalysts, and sensors due to their large
specific surface area and high reactivity (3). The scanning electron microscopy
(SEM) micrographs (Figure2) illustrate the spherical shape of nanoparticles.(4).
Fig 2; Scanning electron micrographs of Polystyrene nanoparticles
Nanoparticles activity:
The activity of gold nanoparticles in addition to their biocompatibility has
made them preferable for ophthalmological implications. Oxidative stress plays
a foremost role in etiology of several diabetic complications. The ability of gold
nanoparticles in inhibiting the lipid from peroxidation thereby preventing the
ROS generation has restored the imbalances in the antioxidants and liver
enzymes responsible for the cell dysfunction and destruction, leading to tissue
injury in the diabetic control group at hyperglycemic conditions so the gold
nanoparticles' regarded as antioxidant(5).
Smaller nanoparticles more strongly favor native-like protein structure,
resulting in higher intrinsic enzyme activity. The mechanism behind the impact
of different sizes of nanoparticles on protein structure can be explained by a
simple model (Figure 5). Larger nanoparticles provide larger surface area of
contact for adsorbed proteins which results in stronger interactions between
proteins and nanoparticles. The greater degree of interaction leads to greater
perturbation of protein structure.(2). a lot of information is available for nanosized silver particles due to their use as bactericides. The cells of bacteria are
damaged in the presence of nano-Ag, finally resulting in death of the organisms
(1)
4
Free
enzyme
enzyme on 4-nm NPs
enzyme on 15-nm NPs
Fig 5. Show the effect of nanoparticles surface area
Enzymes immobilization:
The activity of the enzyme was improved with immobilization on
polystyrene nanoparticles(4). Lipases are an important group of enzymes which
have been widely used in the catalysis of different reactions. These enzymes
have been applied in chemical and pharmaceutical industrial applications due to
their catalytic activity in both hydrolytic and synthetic reactions. However, free
lipases are easily inactivated and difficult to recover for reuse. Therefore,
especially in large-scale applications, lipases are often immobilized on solid
supports in order to facilitate recovery and improve operational stability under a
wide variety of reaction conditions. Some lipase immobilization strategies
involve the conjugation of lipases via covalent attachment, cross-linking,
adsorption and entrapment onto hydrophobic or hydrophilic polymeric and
inorganic matrixes. lipase immobilization technologies is that the activity of
lipases decreases significantly upon immobilization due possibly to changes in
enzyme secondary structure, or limited access of substrate to the active site of
the surface bound enzyme. Lipase immobilization efficiency as well as the
activity of bound enzyme was found to be dependent on conditions used during
preparation of polydopamin magnatic nanoparticles ( PD-MNPs) and
immobilization of lipase. In the absence of polydopamine treatment, binding of
lipase to MNPs was inefficient and resulted in low activity, the PD-MNPs
exhibit high lipase loading capacity due to high surface area and strong
adhesive interactions between lipase and polydopamine. In addition, the
immobilized lipase shows high specific activity and favorable thermal and pH
stability compared to free lipase (6). Recently the anti-glycation activity of gold
nanoparticles in addition to their biocompatibility has made them preferable for
ophthalmological implications(5)
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various nanostructures have been examined as hosts for enzyme
immobilization via approaches including enzyme adsorption, covalent
attachment, enzyme encapsulation and sophisticated combination of these
methods.
In Single enzyme Nanoparticles each enzyme molecule is surrounded
with a porous composite organic/inorganic network of less than a few
nanometers thick. The preparation of SENs represents a new approach that is
distinct from immobilizing enzymes into mesoporous materials or
encapsulating them in sol–gels, (figure 3)
In this unique structure of SENs, the enzyme is attached to the hybrid polymer
network by multiple covalent attachment points. The thickness of the network
around each enzyme molecule is less than a few nanometers. The network is
sufficiently porous to allow substrates to have an easy access to the enzyme
active site (7)
Fig 3. Schematic for SEN synthesis
Glucose oxidase gold nanoparticles (GOD/AuNPs) bioconjugates can
hold the enzyme activity at different pH.( Figure. 4 ) also the thermostability of
as-prepared bioconjugates (curve a) and free enzyme (curve b). The
GOD/AuNPs bioconjugates are more stable against high temperature in contrast
with free enzyme in solution,(8).
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Fig 4. Enzyme pH dependence (A) and thermostability (B) of the GOD/
AuNPs bioconjugates (curve a) and free GOD in solution (curve b)
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Acetylcholinesterase (AChE) is an enzyme that produces choline and an
acetate group by break downing the neurotransmitter acetylcholine. It is mainly
found at neuromuscular junctions and cholinergic synapses in the central
nervous system, where its activity serves to terminate synaptic transmission.
AChE has a very high catalytic activity; each molecule of AChE degrades about
25000 molecules of acetylcholine per second. The produced choline can be back
into the nerve terminals to use in synthesizing of new acetylcholine molecules.
An acetylcholinesterase inhibitor (AChEI) or anti-cholinesterase is a chemical
that inhibits the cholinesterase enzyme from breaking down acetylcholine,
increasing both the level and duration of action of the neurotransmitter
acetylcholine. Monocrotophos is one of the organophosphates which can be as
an AChEI(9).
High local concentration of protein on the nanoparticle surface can also
be used to explain why some nanoparticles inhibit fibril formation: binding
between nanoparticles and amyloid proteins could block active sites for fibril
formation and also lead to low protein concentration in solution, thus causing
inhibition of fibril formation(2)
Mechanism of action:
Heavy metals are toxic and react with proteins, therefore they bind
protein molecules, heavy metals strongly interacts with thiol groups of vital
enzymes and inactivates them. In addition, it is believed that Ag and Au bind to
functional groups of proteins, resulting in protein deactivation and denaturation.
Au and Ag nanoparticles were competitive inhibition for enzyme activity.
Competitive inhibition changed the Km but not the Vmax of the enzyme. gold
nanoparticles colloids can be used in diagnosis and treatment of some kinds of
cancer. Some researches proved that silver nanoparticles colloids are
antibacterial; Therefore, it was useful to know what is the effects of gold and
silver nanoparticles colloids on activities of the different enzymes when enter to
the human body ,then it would be known what the side effects of gold and silver
nanoparticles colloids on the human body is(10). Glutamate Oxaloacetate
Transaminase (GOT) and Glutamate Pyruvate Transaminase (GPT) are
important enzymes which are found in the human body because they are
responsible for the metabolism of amino acids. In view of the importance of
transaminase enzymes reactions like GOT and GPT which form links between
the metabolism of amino acids, carbohydrates and fats. Activation or inhibition
of GOT and GPT by chemicals effects on the metabolism of amino acids,
carbohydrates and fats, this research proved that gold and silver nanoparticles
colloids inhibition of GPT and GOT enzymes; Therefore, catabolism of amino
acids will decrease, then the concentration of amino acids in blood would
increase and cause buildup of protein in blood and effect on urea cycle and
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tricarboxylic acid cycle (10). Nanoparticles were found to inhibit enzyme
activity by interacting with the hydrophobic cavity of certain enzymes and
inducing oxidative stress and thus have been investigated as therapeutic agents
against enteric pathogens. These redox-active nanoparticles may interact with a
variety of essential enzymes related to energy and biosynthesis pathways(11).
In animal models nanosilver alters the expression of matrix metallo-proteinases
(proteolytic enzymes that are important in various inflammatory and repair
processes, suppresses the expression of tumor necrosis factor (TNF)-a,
interleukin (IL)-12, and IL-1b, and induces apoptosis of inflammatory cells.
Moreover, silver nanoparticles (diameter 1499.8 nm) modulate cytokines
involved in wound healing (13).
b-Lactamases (Blas) are a family of bacterial enzymes that can hydrolyze the blactam ring in penicillins and cephalosporins with high catalytic efficiency and
render the bacteria resistant to the b-lactam antimicrobial reagents the use of Bla
to cleave the modified dithiol linker from the cephem nucleus and to induce the
crosslinking of Au NPs, does not require specific instrumentation or
complicated experimental steps. It can offer an alternative platform to evaluate
the enzymatic kinetic reactions and to screen Bla inhibitors in real time (14).
The bactericidal effect of silver ions on micro-organisms is very well known;
however, the bactericidal mechanism is only partially understood.
It has been proposed that ionic silver strongly interacts with thiol groups of
vital enzymes and inactivates them. The present findings, along with reported
interactions of silver nanoparticles with thiol rich enzymes and bacterial
genomic DNA, can explain the inhibitory effect of the nanoparticles on growth
of gram negative bacteria(15). The use of antimicrobial enzymes covalently
attached to nanoparticles is of special interest because of enhanced stability of
protein-nanoparticle conjugates. Colloidal nanoparticles provide an almost ideal
remedy to the usually contradictory issues encountered in the optimization of
immobilized enzymes: minimum diffusional limitation, maximum surface area
per unit mass, and high enzyme loading (16).
Nanotoxicity :
Nanoparticles may interact with proteins when they enter the human
body. However, whether the presence of nanoparticles will disrupt normal
protein function and cause adverse side-effects (2). A consistent body of
evidence shows that nano-sized particles are taken up by a wide variety of
mammalian cell types, are able to cross the cell membrane and become
internalized. The uptake of NP is size-dependent and the aggregation and sizedependent sedimentation onto the cells or diffusion towards the cell were the
main parameters determining uptake(1) The current hypothesized nanotoxicity
mechanisms include suppression of energy metabolism, oxidative damage to
crucial proteins and enzymes, and increased membrane permeability, causing its
rupture(11).
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The nontoxic effect of the gold nanoparticles was confirmed for no sub clinical
toxicology through hematological analysis and histological studies over the vital
organs (liver, kidney, spleen and lung) after the administration of gold
nanoparticles for 15days(5).
In general, two mechanisms are involved in the toxicity of nanoparticles.
One mechanism is that nanoparticles themselves directly exert toxicities, which
are related to the chemical component, size and shape of nanoparticles. When
some nanomaterials gain entry into the body, either via inhalation, dermal or
oral routes, and penetrate into cells, they can subsequently pose a series of
cytotoxicities or promote DNA damage by several mechanisms. For example,
nanoparticles can physically interact with the DNA molecule or proteins, which
may lead to physical damage to the cell or genetic material. In addition,
inflammation and oxidative stress (generation of reactive oxygen species)
induced by nanoparticles have been identified as giving rise to effects on cell
membranes, cytoplasm, nuclei and mitochondrial function. Importantly,
nanoparticles can damage cells through the regulation of redox-sensitive
transcription factors, induction of apoptotic and necrotic cell death and
decreased proliferation, and DNA damage responsive signalling. The other
mechanism of nanoparticle toxicity is that nanoparticles can be used as delivery
carriers. In cancer and gene therapy, nanoparticles can deliver drugs at high
concentrations to the sites of interest, e.g. cancer lesions. It is not difficult to
understand that if the material the nanoparticles carry is not a drug but highly
toxicant, it may cause potential damage to cells (12).
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