CHEM 40.

1 - MIDTERMS REVIEWER  the action of a phosphate buffer against added base or

Discussion: Buffers
 buffer – a solution that resists drastic changes in pH
when small amounts of acid or base are added
o regulates the pH of fluids and tissues of living
organisms within the limits consistent with life
and normal functions
 most if not all biological processes are tightly regulated
by the pH of the medium in which they occur
o the conformation and activity of biomolecules
as well as the concentrations of molecular and
charged species in solution are very sensitive to
pH changes
 metabolic processes continuously alter intracellular H +
concentration, and since optimum activities of many
biomolecules occur within a limited pH range, living
organisms are equipped with remarkably efficient
mechanisms to maintain intracellular H+ concentration
 in humans, these mechanisms include
o the buffer systems of the body
o the action of the kidneys by which acids and
bases are excreted in the urine
o the respiratory mechanism by which the H+
concentration in bodily fluids is regulated by
the rate of carbon dioxide elimination in the
lungs
 buffers are also used in the laboratory to control the pH
of culture media for microorganisms and tissues
acid is show as:
OH-(aq) + H2PO4-(aq)  HPO42-(aq) + H2O
Acid Conjugate Base
OH-(aq) + HPO42-(aq)  H2PO4-(aq) + H2O
Base Conjugate Acid
 when 2 or more buffers are present, the effects are
additive so that the buffering ability occurs over wider
range
 the buffering action of a weak base and its salt is due to
the ability of the base component to neutralize the
added base
o the buffer solutions of weak bases and their
salts are not commonly used because of the
volatility and instability of the bases and the
dependence of their pH on pKw which is
markedly affected by temperature changes
 pseudo buffers – in contrast to the buffering action of
weak acids, that of dilute solutions of a strong acid or
strong base is due to the amphoteric properties of the
solvent, such as water, which can act as either an acid or
base
 some organic buffers with good buffering capacity in the
physiologically important pH range of 6 – 8 are used in
the biochemistry laboratory
o zwitterionic amino acids, mainly N-substituted
taurines, or N-substituted glycines

Buffer Type Example

1. solutions of weak acid HOAc – NaOAc
and its salt (or
conjugate base)
2. solutions of a primary NaH2PO4 – Na2HPO4
salt and a secondary
N-(2-acetamido)-iminodiacetic acid (ADA)
salt
3. solutions of a weak NH3 – NH4Cl
base and its salt (or
conjugate acid)
4. solutions of amino acids and proteins
ampholytes

Buffer Capacity N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid

 buffer action – is the resistance of a buffer solution to a (HEPES)
change in [H+]
 the buffering action of a solution of a weak acid and its
salt is due to the salt component to neutralize any added
acid, and the ability of the acid component to neutralize
any added base
 consider the HOAc-NaOAc buffer solution:
2-(N-morpholino)-ethanesulfonic acid (MES)
OH-(aq) + HOAc(aq)  OAc-(aq) + H2O
Acid Conjugate Base

H+(aq) + OAc-(aq)  HOAc(aq) + H2O

Conjugate Base Acid

Tris-(hydroxymethyl)aminomethane (Tris or THAM)

CHEM 40.1 – Midterms Reviewer 1 /steffigatdula/

N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid
(TES)
Henderson-Hasselbalch Equation
 the pH of a buffer solution made up of known
concentration of a weak acid and its salt can be
calculated using the Henderson-Hasselbalch equation:
[base]
pH = pK a + log
[acid]
mmol base−mmol H+ add.
addition of SA: pH = pK a + log
mmol acid+mmol H+ add.
mmol base+mmol OH− add.
addition of SB: pH = pK a + log
mmol acid−mmol OH− add.
[acid]
pOH = pK b + log
[base]
mmol acid −mmol OH− add.
addition of SB: pOH = pK b + log
mmol base+mmol OH− add.
mmol acid+mmol H+ add.
addition of SA: pOH = pK b + log
mmol base−mmol H+ add.
 from the Henderson-Hasselbalch equation, calculate
the ratio of salt to weak acid required to obtain the
desired pH
 if both acid and salt form as concentrate and/or stock
solutions or as solid, mix the 2 forms in calculated
amounts ad dilute to a volume just less than desire final
volume of the buffer
o verify the pH using a pH meter
o if necessary, adjust the pH by adding a small
amount of 1 M solution of acid or base until the
desired pH is attained, then add distilled water
to the desired final volume
 if only the base (or acid) form is available, you will have
to generate the acid form first
o calculate the amount of base (or acid)
corresponding to the total number of moles of
buffer needed
 mole base (or acid) = moles buffer
 mole base (or acid) = (desired conc. of
buffer) x (desired volume of buffer)
o this is the amount of base (or acid) you will
measure out
o slowly add small amount of strong acid (or
base) to the measured amount of base (or acid)
until the pH desired is attained
o adjust to the required volume by dilution with
distilled water
Activity: Micropipetting – Transferring Minute Volumes

 ionization of the generalized weak acid HA:

HA(aq)  H+(aq) + A-(aq)

Acid Conjugate Base

[A− ]
pH = pK a + log
[HA]
 the pH of a buffer solution is also influenced by the
following factors:
o addition of neutral salts – a change in ionic
strength changes the pH
o dilution – a decrease in ionic strength
decreases the reserve acidity or alkalinity of
the buffer
o temperature – the pKa of a buffer is dependent
on the temperature; the pH of a buffer can
change as a function of temperature
 to be suitable for a particular application, the buffer
must meet the following requirements:
o buffer pH must be within 1 unit of the pKa  mean - average of the values measured from the
value of the acid or the base sample; use a calculator to get this (x)
o buffer components must be appreciable
soluble in water (x1 + x2 + x3 + . . . xn )
x=
o buffer components should not react with each n
other
 standard deviation, s – square root of variance; use a
General Steps in the Preparation of Buffers calculator to get this (xσn-1); same unit
 choose an appropriate buffer system
o the weak acid component should have a pKa 2 ∑ni=1 (xi − x)2
closest to the desired buffer pH to ensure s= √
n−1
maximum buffer capacity

CHEM 40.1 – Midterms Reviewer 2 /steffigatdula/

 relative standard deviation, RSD Albumin
 most abundant protein in egg whites
s  functions include transport of fatty acids, keeping fluid
RSD = x 100
x from leaking out of the circulatory system, pH
maintenance
 range
R = xhighest − xlowest 20 mL of egg whites + 2.0 precipitates other
mL of 1.0M HOAc, unwanted components
 Grubb’s Test dropwise (coagulum)
o used to detect outliers; can only detect 1 outlier per 
data set filter mixture through cell lysis; membrane
o arrange data set from lowest to highest then damp cheesecloth; press disruption;
calculate |xi − x| for both extremes, calculate gexp against the sides of the homogenization
for the value with a higher |xi − x| funnel
o if gtab > gexp then the value is accepted, otherwise it 
is rejected slowly add an equal “salting in”; at low salt
o if gexp is rejected, calculate the new s and x volume of saturated concentrations, the
for the data set with the outlier removed (NH4)2SO4 (ammonium solubility of the protein
o uses n in the table of critical values sulfate) to the filtrate increases slightly; ions from
the salt associate with the
max |xi − x| surface of the protein, this
i=1..n
g=
s  shields those areas from
the solvent molecules
Experiment 1: Extraction and Isolation of Proteins (water)
incubate at room
Extraction  Isolation  Int. Purification  Fin. Purification temperature for about 30
minutes
 extraction 
o starts with cell lysis centrifuge the mixture and separation based on
o mechanical and non-mechanical cell discard the precipitate density (heavier particles at
disruption  the bottom)
 sonication, French press, mortar and transfer the supernatant
pestle into an Erlenmeyer flask
o additional reagents can be added to aid immersed in an ice bath
extraction 
 suspension in hypotonic buffer add an equal volume of “salting out”; at higher salt
(causes cells to burst) saturated (NH4)2SO4 until concentrations, the
 detergents (may denature proteins) turbidity persists solubility of the proteins
 addition of Glass Beads decrease; all the binding
 addition of Nucleases sites on the protein surface
 lysozyme (dissolves cell walls) for the salt ions have
o addition of protease inhibitors ensure that the become occupied and so
desired proteins will not be hydrolyzed the ions begin to interact
 isolation with the solvent;
o differential solubility separation techniques concentration of "free"
 salting in/out, isoelectric solvent molecules
precipitation, precipitation by decreases as they are used
acetone or alcohols  to solvate the salt ions;
 intermediate purification protein molecules
o removes most of the bulk impurities (i.e. other therefore move closer
proteins and nucleic acids) together and begin to
o IEC, GFC, affinity chromatography, and interact with one another
electrophoretic methods via the hydrophobic or
 final purification charged patches on their
o a highly pure protein is achieved by removing surfaces; at some salt
trace impurities and other closely related concentration , the protein
species molecules aggregate and
o HPLC, isoelectric focusing, 2D-SDS-PAGE come out of solution.
allow mixture to stand for
15 minutes


CHEM 40.1 – Midterms Reviewer 3 /steffigatdula/

 centrifuge the mixture; o absorption at 545 nm is directly related to the
collect precipitate concentration of the protein
 o requires a protein standard and creation of
dissolve with 10 mL 0.9% calibration curve
NaCl; calculate for % w/v  the protein standard should match
the structure and reactivity of the
extracted protein
Casein
 contains all the essential amino acids Advantages Disadvantages
 precipitated thru isoelectric precipitation (pI = 4.7) simple and sensitive, few formation of protein-dye
o CaCaseinate(aq) + 2H+(aq) → Casein(s) + Ca2+(aq) interferences complex can be affected by
high sensitivity (~1 μg) the number of
15 mL of milk + 0.1M HCl isoelectric focusing; casein available/free amino acids
until flocculatent precipitates out within the protein, so
precipitate forms choosing the protein
 standard is important
centrifuge mixture; discard
supernatant  Biuret Assay
 o colorimetric determination of protein
add 1 mL 95% ethanol and removes adsorbed water concentration, measures the number of
invert tube to wash peptide bonds
residue; decant alcohol; o biuret reagent is made in basic solution using
repeat twice copper sulfate (CuSO4) and potassium tartrate
 (K2C4H4O6)
wash precipitate with 1 mL removes adsorbed lipids o the atoms involved in the peptide bond form a
acetone; decant washings complex with Cu2+, forming a violet solution
and air dry under hood that can be measured at 550 nm

dissolve precipitate in 10 Advantages Disadvantages
mL 0.01M NaOH; calculate not significantly affected by lack sensitivity, unsuitable
for % w/v differences in amino acid for solutions with protein
composition between content of less than 1
Experiment 2: Protein Quantitation Through proteins, easily detects the mg/mL
Spectrophotometric Methods presence of peptide bonds

 Warburg-Christian Assay
o estimates protein concentration based on the  Lowry Method
absorbance of exposed tyrosine and o Biuret Assay + Tyr and Trp oxidization by Folin
tryptophan residue and Ciocalteau reagent producing a blue-
o absorbance measured at 280 nm at 260 nm; purple complex
260 nm adjusts for the absorbance of nucleic o extremely sensitive (~1 μg/mL) but is subject to
acids (whose absorption spectra overlap with interferences from a wide range of non-protein
those of tyrosine and tryptophan) substances
o protein conc. (mg/mL) = 1.5(A280)-0.76(A260) o requires external calibration
o ratio of A280/A260 can estimate the purity; o incubation increases sensitivity
A280/A260 = ~1.75, the higher the % purity
 Bicinchoninic Assay (BCA)
o purple colored product (λmax = 562 nm)
Advantages Disadvantages o more sensitive than Biuret and Lowry, less
simple, non-destructive, only qualitative/semi- variability than Bradford
interfering substances can quantitative o incubation increases sensitivity
be accounted will account only for the o high sensitivity; 1 μg/mL
moderate sensitivity (50- free aromatic amino acids  Ninhydrin Method
1000 μg) in the protein o determines the amount of free amino nitrogen
o yellow  deep purple product
 Kjeldahl Method
 Bradford Assay o used to estimate protein content in foods and
o the binding of Coomassie Brilliant Blue G-250 other samples
to protein under acidic conditions causes a shift o quantitative determination of total N = sum of
in the dye’s wavelength of maximum organic N, ammonia and ammonium
absorption from 465 nm (brown) to 595 nm
(blue)

CHEM 40.1 – Midterms Reviewer 4 /steffigatdula/

Experiment 3: Monitoring Protein Conformational Changes
by Viscosity and CD Spectroscopy Experiment 7: Enzyme Kinetics

Denaturants

Denaturant Factor Molecular Level

HCl pH Destroys H bonds,
affects electrostatic
NaOH pH interactions

Heat Temperature Destroys weak

interaxns, can
irreversibly denature
protein
NaCl Ionic Affects ion bridges;
Strength leads to solvation

Urea Chaotropic Denatures protein by

Agent allowing water to
solvate hydrophobic
groups
SDS Detergents Amphiphatic, tail
disrupts hydrophobic
interaxns

β-MeOH Reducing Converts disulfide

Agent bridges to sulfuhydril
groups

Viscosity
 observes alterations in the tertiary and quaternary
structures of proteins
 viscosity – measure of a liquid’s resistance to flow
o denaturation causes protein (albumin) to go
from globular to rod-shaped/extended form;
viscosity goes up
o denaturation of globular proteins causes
unfolding of compact tertiary structures into
flexible stretched-out amino acid chains


CD-ORD

Experiment 4: Ion Exchange Chromatography

Experiment 5: Protein Characterization by Gel Filtration

Chromatography

Experiment 6: Protein Characterization by Electrophoresis

CHEM 40.1 – Midterms Reviewer 5 /steffigatdula/