Spectrophotometry

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Spectrophotometry
Enzymes
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An absorbance spectrophotometer is an instrument
that measures the fraction of the incident light
transmitted through a solution
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What does that mean?
In other words, it is used to measure the amount
of light that passes through a sample material
and, by comparison to the intensity of light
passing through a reference blank, they
indirectly measure the amount of light absorbed
by that sample
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Spectrophotometers are designed to transmit light
of narrow wavelength ranges. A given compound
will not absorb all wavelengths equallythats
why substances are of different colors
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Because different compounds absorb light at
different wavelengths, a spectrophotometer can be
used to distinguish compounds by analyzing the
pattern of wavelengths absorbed by a given sample
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Also, the amount of light absorbed is directly
proportional to the concentration of absorbing
compounds in that sample, so a spectrophotometer
can also be used to determine concentrations of
compounds in solution
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When studying a compound in solution by
spectrophotometry, you put the solution in a
sample holder called a cuvette and place it in
the spectrophotometer
Light of a particular wavelength passes through
the solution inside the cuvette
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Because other compounds in a solution may absorb
the same wavelengths as the compound being
analyzed, we compare the absorbance of our test
solution to a reference blank
The reference blank should contain everything
found in the sample solution
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except the substance you are trying to analyze
or measure
We use the reference blank to zero out the
machine so that the absorbance of the reference
blank is set at zero
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Once we place the cuvette in the machine, the
amount of light transmitted or absorbed by the
solution is measured by a light meter. The amount
of light transmitted through the solution is
referred to as transmittance (T)
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The transmittance is defined as the ratio of the
light energy transmitted through the sample to
the energy transmitted through the reference blank
Light transmitted through the sample
Light transmitted through the reference blank
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The absorbance is the amount of light absorbed by
the solution, and is related logarithmically to
transmission
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What this means is that the light received at the
phototube is measured as the percent
transmittance (T) or the log of its inverse,
absorbance (A)
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See the example on page 88 of the lab manual,
under the heading Part 3
Make sure you understand this example!
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An absorbance spectrum is a plot of absorbance as
a function of the wavelength of light, and is
determined to select the optimal wavelength for
analyzing a given compound
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The optimal wavelength for measuring absorbance
is that wavelength that is most absorbed by the
compound in question
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This parameter provides maximum sensitivity for
your measurements
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Enzymes
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Enzymes The groove on the left side of the
enzyme is the active sitethe small red molecule
is the substrate on which the enzyme acts
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• A catalyst is a chemical agent that changes the
  rate of a reaction without being consumed by the
  reaction.
• An enzyme is a catalytic protein.
• Enzymes regulate the movement of molecules
  through metabolic pathways.

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Chemical reactions between molecules involve both
bond breaking and bond forming. To hydrolyze
sucrose, the bond between glucose and fructose
must be broken and then new bonds formed with a
hydrogen ion and hydroxyl group from water.
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• Even in an exergonic reaction, the reactants must
  absorb energy from their surroundings, the free
  energy of activation or activation energy (EA),
  to break the bonds.
• This energy makes the reactants unstable,
  increases the speed of the reactant molecules,
  and creates more powerful collisions.
• In exergonic reactions, not only is the
  activation energy released back to the
  surroundings, but even more energy is released
  with the formation of new bonds.

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Activation energy is the amount of energy
necessary to push the reactants over an energy
barrier.
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• For some processes, the barrier is not high and
  the thermal energy provided by room temperature
  is sufficient to reach the transition state.
• In most cases, EA is higher and a significant
  input of energy is required.
• A spark plug provides the energy to energize
  gasoline.
• Without activation energy, the hydrocarbons of
  gasoline are too stable to react with oxygen.

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Enzymes speed reactions by lowering energy of
activation The transition state can then be
reached even at moderate temperatures.
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A substrate is a reactant which binds to an
enzyme. When a substrate or substrates binds to
an enzyme, the enzyme catalyzes the conversion of
the substrate to the product. Sucrase is an
enzyme that binds to sucrose and breaks the
disaccharide into fructose and glucose.
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The active site of an enzymes is typically a
pocket or groove on the surface of the protein
into which the substrate fits. The specificity of
an enzyme is due to the fit between the active
site and that of the substrate. As the substrate
binds, the enzyme changes shape leading to a
tighter induced fit, bringing chemical groups in
position to catalyze the reaction.
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The rate that a specific number of enzymes
converts substrates to products depends in part
on substrate concentrations. At low substrate
concentrations, an increase in substrate speeds
binding to available active sites. However, there
is a limit to how fast a reaction can occur.
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At some substrate concentrations, the active
sites on all enzymes are engaged, called enzyme
saturation. The only way to increase productivity
at this point is to add more enzyme molecules.
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Temperature has a major impact on reaction
rate. As temperature increases, collisions
between substrates and active sites occur more
frequently as molecules move faster.
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However, at some point thermal agitation begins
to disrupt the weak bonds that stabilize the
proteins active conformation and the protein
denatures. Each enzyme has an optimal temperature.
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Temperatures that are too high will cause the
enzyme (protein) to denature
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Because pH also influences shape and therefore
reaction rate, each enzyme has an optimal pH
too. This falls between pH 6 - 8 for most enzymes.
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When optimal pH is exceeded, the enzyme will also
denature. This, of course, renders the enzyme
non-functional
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There is a limit to the amount of substrate that
a single enzyme molecule can process in a given
time. If the concentration of substrate is
increased, the rate at which product is formed
also increases, up to a maximum value
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At that point the enzyme molecule is saturated
with substrate, and the rate of reaction (Vmax)
depends only on how rapidly the enzyme can
process the substrate molecule.
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The other kinetic parameter frequently used to
characterize an enzyme is its Km, the
concentration of substrate that allows the
reaction to proceed at one-half its maximum rate
(0.5 Vmax)
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A low Km value means that the enzyme reaches its
maximum catalytic rate at a low concentration of
substrate and generally indicates that the enzyme
binds to its substrate very tightly, whereas a
high Km value corresponds to weak binding.
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At Vmax, all substrate-binding sites on the
enzyme molecules are fully occupied
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Web Resources
National Center for Biotechnology Information
(NCBI)
http//www.ncbi.nlm.nih.gov/