Introduction To Biotech Instrumentation: Basic biochemical methods

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Introduction To Biotech Instrumentation Basic
biochemical methods

• By
• Yao-Te Huang
• Sep 24, 2009

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Lecture Outline

• Ionization of water, weak acid weak base
• Buffer preparation measurements of pH
• Dialysis, filtration, concentration
• lyophilization
• Protein quantification
• Radioisotopes in biochemistry

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Ionization of water, weak acids, and weak bases
(1)
Pure water is slightly ionized.
Kw (the ion product) for water At 25 C
At the neutral pH, HOH-
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Ionization of water, weak acids, and weak bases
(2)
The pH scale designates the H and OH-
concentrations
alkaline or basic OH- gt H in general or
solutions having a pH greater than 7 at 25C
acidic H gtOH- in general
or solutions having a pH less than 7 at 25C
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Ionization of water, weak acids, and weak bases
(3)
Weak acids and bases have characteristic
dissociation constants
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Titration curves reveal the pKa of weak acids (1)
Titration curve a plot of pH against the amount
of NaOH added
At the midpoint, pHpKa CH3COOH CH3COO-
The buffering region pKa/- 1
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Titration curves reveal the pKa of weak acids (2)
Three weak acids acetic acid, dihydrogen
phosphate, and ammonium ion
The stronger the acid, the lower its pKa
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Amino acids have characteristic titration curves
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Titration curves predict the electric charge of
amino acids
pI (isoelectric point or isoelectric pH) The
characteristic pH at which the net electric
charge is zero
At any pH gt pI, the molecule has a net negative
charge At any pH lt pI, the molecule has a net
positive charge
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Amino acids differ in their acid-base properties
pI1/2 ( 6.09.17) 7.585
pI ½ (2.194.25) 3.22
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pKa values of 20 common Amino acids
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Buffers are mixtures of weak acids and their
conjugate bases
Buffer something that resists large changes in
pH when adding small amounts of H (acid) or OH-
(base)
Take the acetic acid-acetate pair for
example The titration curve of acetic acid has
a relatively flat zone extending about 1 pH unit
on either side of its midpoint pH of 4.76
(pKa). This relatively flat zone is the
buffering region of the acetic acidacetate
buffer pair. At the midpoint of the buffering
region, where the concentration of the proton
donor (acetic acid) exactly equals that of the
proton acceptor (acetate), the buffering power of
the system is maximal.
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The Henderson-Hasselbalch equation relates pH,
pKa, and buffer concentration
The Henderson-Hasselbalch equation
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Buffer preparation

• Weight electric balance (mg to 100g)
• Volume graduated cylinder (large vol.) pipet
  (ml to 10-20 ml)
• micropipet (0.1-1 ?l to several ml)

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Buffer Preparation

• The effective range of a buffer system pKa ? 1
• Some common buffers
• Phosphate buffer (pKa1.966.7012.32)
• Acetate buffer (4.76)
• Tris buffer (8.08)
• ionic strength

Mi molarity of the ion Zi the net charge of
the ion
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pH meter an instrument that measures the pH
value of the unknown

• pH meter obtains the pH value of an unknown by
  measuring the very small potential difference
  in the circuit

V voltage of the completed circuit Eref
potential of reference electrode
Calibration of pH meter using pH 4, pH 7, pH
10 buffer standard
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Dialysis used for buffer exchanges or for
removing small molecules

• (1) sealing a solution containing both
  macromolecules and small molecule impurity (such
  as (NH4)2SO4) in membrane tubing (bag).
• (2) placed a sealed bag in a large container
  of low ionic strength buffer with stirring at
  least one-time fresh buffer replacement.
• (3) Time after 4-6 hours or longer.
• (4) Temperature 4 ºC.

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Dialysis (contd.)

• (5) leaving a small air bubble (about 10 of
  the total volume) in the dialysis bag (to allow
  the bag to float)
• (6) materials e.g., cellulose (MW cut-off 1
  to 50 KD). Dialysis tubing has small pores in it
  (i.e., it is semi-permeable) which permit passage
  of the small inorganic salt molecules but not the
  macromolecular proteins.
• (7) tubing is contaminated with glycerol (for
  removing, immersing 1 acetic acid for 1 hr),
  heavy metal ions (for removing, boiling in EDTA
  solution for 1 hr)
• (8) storage of dialysis tubing in distilled
  water with some NaN3 several drops of
  chloroform

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Dialysis (contd.)
As the small molecules diffuse out of the bag and
into the dialysis buffer, the ionic strength of
the protein solution decreases. The process
continues until the salt concentrations inside
and outside the bag are identical.
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filtration

• filters depth filters (having a high load
  capacity, ill-defined pore sizes, trapping
  particles both on the surface and within the
  matrix) screen filters (having uniform pore
  size, retaining particles on the surface)
• Microfiltration 0.22?m (bacteria)
• 0.45 ?m (other bigger
  particles)
• Concentration of protein solution Amicon (by
  applying high pressure), or Centricon (by
  applying the centrifugal force)

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filtration
(filter)
(held by a spring clamp)
(water aspirator)
Amicon
Suction filtration
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The Amicon concentrator A membrane disk is
placed in a pressure cell, and the sample
solution is placed on the top of it. The sample
port is closed and pressure (10 to 80 psi) is
exerted on the liquid surface using compressed
nitrogen.
centricon
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Lyophilization (freeze-drying)

• Involved in the removing of solvent from a frozen
  sample.
• Lyophilizer an instrument (consisting of a
  refrigeration unit a vacuum pump) doing
  lyophilization at low temperature ( -40º C)
  low pressure (vacuum)

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Lyophilization (freeze-drying)
Lyophilizer
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Protein quantification (The Biuret assay)

• Principle peptide (N atom) bonds alkaline Cu2
  
• -gt purple complex measure A540
• Sensitivity low (1-20 mg)
• Interferences Goods buffers, Tris some amino
  acid buffers
• Time moderate, 20-30 min

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Protein quantification (The Lowry assay)

• Principle (1) Biuret reaction (2) reduction of
  phosphomolybdate-phosphotungstate by Tyr Trp
• Sensitivity High (5?g)
• Interferences Goods buffers (e.g., MOPS),
  ammonium sulfate, glycine, mercaptans
• Time slow, 40-60 min

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Protein quantification (The Bradford assay)

• Principle (1)?max of Coomassie Brilliant Blue
  dye changes from 465 nm to 595nm upon binding to
  protein (2) measure A595
• Sensitivity High (5?g)
• Interferences detergents, Triton X-100, SDS
• Time rapid, 15 min

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Protein quantification (The spectrophotometric
assay)

• The first three assays (1) involves colored
  product formation (2) need to do standard curve
  using (e.g., BSA (Bovine Serum Albumin))
• The current assay
• Principle absorbance of 280 nm light by Trp
  Tyr residues in protein
• Time rapid, 5-10 min
• Sensitivity moderate sensitivity, 50-100 ?g
• Interferences nucleic acids, purines,
  pyrimidines

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Radioisotopes in biochemistry

• Isotopes with the same number of protons, but
  different number of neutrons
• (e.g. hydrogen (1H1), deuterium (1H2),
  tritium (1H3)
• Radioisotopes isotopes that undergo spontaneous
  nuclear disintegration accompanied by emission of
  particles
• Three types of emission of particles
• ? particle (a He nucleus), ß particle (a
  high-energy electron), ? ray (very high- energy
  photon)

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Radioisotopes in biochemistry

• An equation of radioactive decay

Where ? is the decay constant
gt
where N0 is N at t0
The half-life (t1/2) the time required for 50
of the radioactive atoms to decay
t1/2 ? ? 0.693
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Derivation of
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Derivation of
(contd.)
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Commonly used radioisotopes (1) a rough view

• P32 15P32 -gt -1ß0 16S32
• (t1/214.2 days)
• S35 16S35 -gt -1ß0 17Cl35
• (t1/287.1 days)
• H3 1H3 -gt -1ß0 2He3
• (t1/212.3 years)
• C14 6C14 -gt -1ß0 7N14
• (t1/25570 years)

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Commonly used radioisotopes (2) a detailed view

• P32 15P32 -gt -1ß0 16S32
• (t1/214.2 days)
• S35 16S35 -gt -1ß0 17Cl35
• (t1/287.1 days)
• H3 1H3 -gt -1ß0 2He3
• (t1/212.3 years)
• C14 6C14 -gt -1ß0 7N14
• (t1/25570 years)

The conservation of the lepton number
Anti-electron neutrino
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Radioisotopes in biochemistry Unit of
radioactivity

• One Curie (Ci) the amount of radioactive
  material that emits particles at a rate of
  3.7?1010 disintegrations per second (dps) or
  2.2?1012 min-1 (dpm)
• Millicurie (mCi10-3 Ci) microcurie (?Ci10-6
  Ci)
• Specific activity the amount of radioactivity
  per unit mass of the material
• cpm (counts per minutes)
• cpmdpm ? efficiency of counting device

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Liquid scintillation counting

• The sample is dissolved or suspended in the
  scintillation cocktail. The emitted ßparticle
  causes a pulse of light (through a series of
  reactions), which is detected by PMT
  (photomultiplier tube), and amplified and
  registered by a scaler (a counting device)

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Liquid scintillation counting
The light of longer wavelength (400-470 nm) is
more efficiently detected by a PMT
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Liquid scintillation counting
A coincidence circuit counts only those flashes
that arrive Simultaneously at the two PMTs.
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Liquid scintillation counting
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Liquid scintillation counting
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Autoradiography

• In which the sample containing the radioactive
  substance is placed near a photographic emulsion.
  Radiation emitting from the sample activates
  silver halide that is further converted into
  metallic silver during the film development
• for localization (or quantitative) studies of
  radio-labeled biomolecules

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Autoradiography (contd.)
A radiation-sensitive photographic emulsion
containing silver salts (AgBr) is placed over
tritium-labeled cells attached to a glass slide
(for the light microscope) or to a carbon-coated
grid (for the electron microscope). The cell
regions containing the labeled molecules emit
radioactive particles, along the tracks of which
silver is deposited. When the photographic
emulsion is developed, the silver deposits appear
as dark grains under the light microscope and as
curly filaments in the electron microscope.