The Henderson-Hasselbach Equation 2.

The acid component (H2CO3) is regulated by pulmonary

ventilation.
•Derived from the formula for dissociation constant of an acid, Ka:
Applications Of The Henderson Hasselbalch Equation
Ka = [A-][H+]/[HA]
•Carbonic acid is both the major acid and the buffer produced by the
•Taking the -log of both sides gives us: body.
-log Ka = -log {[A-][H+]/[HA]} •The pKa of carbonic acid is 3.8. The pH of blood is 7.4. Carbonic
acid completely disassociates in blood and is theoretically unable to
•The left side is the definition of pKa:
buffer and generate bicarbonate.
pKa = -log {[A-][H+]/[HA]}
•Carbonic acid can be replenished from CO2 found in body fluids
•The right side can be re-written using the log rules: pKa = -log [A-] - and air because the concentration of dissolved CO2 in body fluids is
log [H+] - (-log [HA]) about 500 times greater than that of Carbonic acid.

•The second right side term is the definition of pH: pKa = -log [A-] + •A buffer can be created by mixing a weak acid (e.g. acetic acid) with
pH + log [HA] its conjugate base (acetate)

•Solving for pH: •If HCl (acid) is then added to the solution, acetate can neutralize it,
and be converted to acetic acid in the process.
pH = pKa + log [A-] - log [HA]
•If a base is added, acetic acid can neutralize it, and be converted to
•Using the log rules in reverse gives us the equation as it is normally acetate in the process
written: pH = pKa + log [A-]/[HA]
•Physiologic buffers include bicarbonate, inorganic
✓ Where pKa is the negative logarithm of the acid’s orthophosphates, and proteins.
dissociation constant

PHOSPHATE BUFFER
Applications Of The Henderson Hasselbalch Equation
•It is the major intracellular buffer. Its pK’ value of 6.86 is near the
•The behavior of weak acids and buffers is described by the intracellular pH of 7.0. It consists of the following components:
Henderson-Hasselbalch equation.

•The pH of a solution containing a weak acid is related to its acid

PROTEINS
dissociation constant.
•They may act as buffers because many of them have amino acids
•Solutions of weak acids and their conjugate bases (or of weak bases
which behave like weak acids. For example, histidine acts as a buffer
and their conjugate acids) exhibit a buffering system.
because of presence of imidazole group
•Important physiologic buffer systems include bicarbonate,
•It has a pK’ value of 6.0 which is close to the physiological pH.
inorganic orthophosphates, and intracellular proteins such as
hemoglobin. •The respiratory center within the hypothalamus which controls the
rate of breathing is sensitive to changes in pH.
•The normal pH range of arterial blood is from 7.35 to 7.45.
•As the pH falls, individuals breathe more rapidly and expire more
•The kidneys and the lungs also play major roles in maintaining pH.
CO2. As the pH rises, they breathe more shallowly.
•Dissolved CO2 is in equilibrium with the CO2 in the air in the alveoli
•Thus, the rate of breathing contributes to regulation of pH through
of the lungs, thus the availability of CO2 can be increased or
its effects on the dissolved CO2 content of the blood.
decreased by an adjustment in the rate of breathing and the amount
of CO2 expired.

•The pKa for dissociation of bicarbonate anion (HCO3) into H and Blood pH Regulation
carbonate (CO32-) is 9.8; therefore, only trace amounts of carbonate
exist in body fluids. •Done by maintenance of Carbon Dioxide (CO2) and Bicarbonate ion
concentration
BUFFER
BICARBONATE BUFFER
ACIDS IN THE BLOOD OF A HEALTHY PERSON (HCO3)
It is the principal extracellular buffer, comprising carbonic acid (the
proton donor) and bicarbonate (the proton acceptor). •CO2 is an acid, HCO3- is its conjugate base

•It functions in the same way as other conjugate acid-base pairs. •The pKa for this system is 6.1
However, there are important differences:
•CO2 level is regulated by the lungs
1.The base constituent, bicarbonate ( 3 HCO-) is regulated by
•HCO3- level is regulated by the kidneys
kidneys.
 4.Correction of metabolic acidosis consists of increased excretion of
the excess fixed H+ as titratable acid and NH4+, and increased
ACIDS IN THE BLOOD OF A HEALTHY PERSON reabsorption of “new” HCO3-, which replenishes blood HCO3-
concentration
Clinical Application

•When, excessive production of acids is the cause of metabolic

METABOLIC ACIDOSIS
acidosis, concentration of bicarbonate decreases but that of Cl
• Results from a primary fall in bicarbonate level (<24 remains unaffected.
mmol/L) resulting in decreased pH of arterial plasma (because of
•In renal tubular acidosis or acetazolamide therapy (an inhibitor of
decreased ratio for bicarbonate concentration to pCO2 in the
carbonic anhydrase), fall of bicarbonate is accompanied by an
Henderson-Hasselbalch equation
increase in chloride ion concentration. Hence, anion gap does not
Causes: change in these conditions, which are called normal anion gap
acidosis or hyperchloremic acidosis.
✓ Ingestion of drugs or toxic substances

▪ Methanol
ANION GAP ACIDOSIS
▪ Ethanol
•Anion gap is estimated by measuring the differences between the
▪ Salicylates sums of the concentrations of principal cations (Na+ and K+) and
principal anions (Cl- and 3 HCO-)
▪ Ethylene glycol
•Average reference values of these ions are:
▪ Ammonium chloride
•Therefore, in healthy individuals the anion gap has an average
Loss of bicarbonate ions value of 19mmol/L.
▪ Diarrhea

▪ Pancreatic Fistulas RESPIRATORY ACIDOSIS (S)

▪ Renal Dysfunction •It results from a decrease in alveolar ventilation, causing decreased
elimination of CO2 by the lungs.

•The consequent increase in PCO2 is the primary event.

Lactic Acidosis
•Can occur from pathological conditions that damage the
▪ Hypoxemia
respiratory centers or that decrease the ability of the lungs to
▪ Anemia, carbon monoxide eliminate CO2.

▪ Shock (hypovolemic, cardiogenic, septic, etc.)

▪ Severe exercise Compensation For Respiratory Acidosis

▪ Acute respiratory distress syndrome (ARDS) •In contrast to metabolic acidosis, where respiratory compensation
is important, in this type of acidosis the primary defect in lungs, the
Ketoacidosis physiological response of lungs cannot be expected to play any
significant role.
▪ Diabetes mellitus
•Renal response plays an important role in bringing the acid-base
▪ Alcoholism status back to normal.
▪ Starvation The kidney responds to acidosis by actively secreting fixed acids
Inability to excrete hydrogen ions while retaining filtered HCO3

▪ Renal dysfunction

1. Overproduction or ingestion of fixed acid or loss of bases

produces a decrease in arterial (HCO3). This decrease is the primary
disturbance of metabolic acidosis.

2. Decreased HCO3- concentration cases a decrease in blood

pH (Acidemia).

3. Acidemia causes HYPERVENTILATION, which is the

respiratory compensation for metabolic acidosis
Amino Acids → Niacin, serotonin, melatonin

•Protein Architecture – alphabet or building blocks of proteins → Histamine

•20 amino acids in mammalian system → GABA

→ Although more than 300 amino acids occur in Structural

nature, proteins are synthesized almost exclusively from the set of → collagen, prothrombin, desmosine, myosin
20 L-α-amino acids encoded by nucleotide triplets called codons
Source of energy:
•ALL proteins are LINEAR polymers of amino acids
→ plays major role in activation / inactivation of enzymes via
•Amino acids have a carboxyl group and amino group are H-bonded phosphorylation
bonded to the same alpha-carbon atom
Chemical messengers

→ GABA (γ-Aminobutyrate) – inhibitory neurotransmitter by

Functions Of Amino Acids altering transmembrane potential differences

Amino Acids performs or pre-empts a dynamic set of functions Metabolic Intermediates

within the body.
→ Ornithine, Citrulline – intermediate in urea synthesis
→ Enzymatic Catalysis
→ Homocysteine, Homoserine, and glutamate-γ-
→ Transport and Storage semialdehyde – intermediary metabolism of protein AA

→ Muscle Contraction → Phenylalanine, Tyrosine – serve as precursors of

epinephrine, norepinephrine, and DOPA (dihydroxyphenylalanine)
→ Immune Protection

→ Generation and Impulse Transmission

Properties Of Amino Acids

1. Based on Central Carbon

•Identify the different characteristics of amino acids.
Optical activity
•Describe the structure of amino acids and peptide bonds.
→ consequence of chirality
•Explain the concept of H+ dissociation in amino acids and its
consequences. → ability of molecules to rotate plane of polarized light ▪
Dextrorotatory (d) –clockwise (right)
•Discuss the different levels of protein structure.
o D-amino acids–bacterial cell w all & some antibiotics
•Name some peptides of physiologic importance and their
functions. o D-aspartate & D-serine –found in human brain ▪ Levorotatory (l) –
counterclockwise (left)

→ Enantiomers –rotate light the same number of degrees

Amino Acids
2. Based UV Absorption
•They differ from each other in their side chains or R groups

→ Responsible for specific function, properties, size/

structure, polarity/ charge, and which influence its solubility in water 1. Based on Central Carbon

•Alpha-Carbon of all amino acids is chiral or asymmetric except Asymmetry or chirality

glycine.
• Chiral center (α carbon)- lends asymmetry to amino acids
→ Alpha-carbon is bonded to 4 different groups. ▪ Chiral
Carbon – carbon in which all four different groups are attached. → Exception: Glycine –not chiral (Achiral)

→ Confers optical activity Chirality- same as enantiomers (2 stereoisomers that are non-
superimposable mirror images)
▪ All molecules with a chiral center are optically active – that is, they
rotate plane-polarized light. • Existence of enantiomers/ mirror image isomers that
cannot be superimposed
Precursor of :
• Either D or L isomers
→ Heme, purine, creatine, glutathione, hippuric acid

→ Epinephrine, melanin
L-α-Amino Acids
•Absolute configuration of amino acids is referenced to • Hydrophobic, oily/ lipid-like
Glyceraldehyde
→ sidechains of nonpolar AA cluster together in the interior
•All amino acids derived from natural proteins are of the L- of the protein
configuration
▪ AA with aliphatic or aromatic R group are hydrophobic and
•Both D-amino acids and non–α-amino acids occur in nature, but occurs in the interior of proteins
proteins are synthesized using only L-α-amino acid
• Does not donate or accept H+ (protons)

• Sidechains do not participate in H-bonding or ionic

3. Acid-Base Property bonding

•the amino and carboxylic acid groups readily ionize • Proline –forms a ring structure due to its imino group

•pK (α-COOH) = pH 2.2 2.ALIPHATIC SIDE CHAIN

•pK (α-NH2) = pH 9.4

•Amphoteric: ability of an amino acid to act as a base (NH2) and an 1. Glycine (G)
acid (COOH)
→ simplest AA: R group is just H atom

→ smallest AA
Different Forms Of Amino Acids
→ Smallest amino acid
1. Unionized form
• Can fit in regions inaccessible to other amino acids
2. Dipolar ion or zwitterion form
• it often occurs where peptides bend sharply
•→ Amino acids exist as zwitterions, a dipolar ion that results from
Methionine (M)
an internal acid-base reaction
•source of SAM (S-adenosylmethionine) active methyl (CH3) donor
•→ Note that the net charge of the zwitterion is zero
in the body
3. Fully protonated [pH 1]

4. Fully ionized [pH 11]

Polar, Uncharged Amino Acids [STCYQN]

1.Hydroxyl-containing AA
Nonpolar Amino Acids [GAVLIPMWF]
→ S,T,Y
1.AROMATIC SIDE CHAIN
✓ can participate in H-bonding and can be phosphorylated

Tryptophan Phenylalanine
2.Amide derivatives of Aspartate (D) and Glutamate (E)
(W) = Indole (F) = Benzene
• have a carbonyl group C=O and amide group
ring ring
• can also participate in H-bonding

• Important in detoxification of ammonia

Classification Of Amino Acids • Glutamine is a derivative of glutamate; primary source of
I. Based on (R) group’s polarity urinary ammonia

A. Nonpolar • Asparagine is a derivative of aspartic acid

B. Polar (no charge)

C. Polar (charged/ ionic) 3.Sulfhydryl-containing AA

II. Based on Synthesis • Thiol (-SH) group

A. Essential • Polar

B. Non-essential • Capable of H-bonding

• Essential- cannot be synthesized by the body. As a result,

these amino acids should be taken up from the diet Polar, Charged, Acidic [DE]
• Proton donors •Necessary for tissue growth and the absorption of zinc and
selenium, minerals that are vital to your health
• At physiologic pH, they are negatively charged and called
aspartate or glutamate Leucine:

•branched-chain amino acid that is critical for protein synthesis and

muscle repair.
Selenocysteine
•Helps regulate blood sugar levels, stimulates wound healing and
✓ Referred to 21st amino acid produces growth hormones

✓ Selenium atom replaces the sulfur of its structural analog, Threonine:

cysteine
•principal part of structural proteins such as collagen and elastin,
which are important components of the skin and connective tissue.

Polar, Charged, Basic [HKR] •Plays a role in fat metabolism and immune function

• Proton acceptors Tryptophan:

• At physiologic Ph, they are positively charged •Often associated with causing drowsiness, tryptophan has many
other functions.
• Histidine
•Needed to maintain proper nitrogen balance
✓ is needed for the buffering capacity of HgB
•a precursor to serotonin, a neurotransmitter that regulates your
✓ involved in the synthesis of histamines appetite, sleep and mood

✓ has aromatic IMIDAZOLE group Isoleucine:

• Lysine has a 2nd primary group •involved in muscle metabolism and is heavily concentrated in
muscle tissue.
• Arginine has GUANIDINO group
•Important for immune function, hemoglobin production and
Types Of Interaction energy regulation
1. Hydrophobic - for nonpolar R-groups (GAVLIPMFW) Lysine:
2. Disulfide bond - Between two sulfur atoms •protein synthesis, hormone and enzyme production and the
absorption of calcium.
Cysteine + Cysteine = Cystine
•important for energy production, immune function and the
3. H-bonding - all amino acids except nonpolar R-groups
production of collagen and elastin
(polar AA)
Histidine:
4. Ionic interaction - between charged AA (D,E, and H, K, R)
•Used to produce histamine, a neurotransmitter that is vital to
5. -OH of serine and-SH of Cysteine - nucleophiles, function
immune response, digestion, sexual function and sleep-wake cycles.
as such during enzymatic catalysis
•Crucial for maintaining the myelin sheath, a protective barrier that
6. -OH pf serine, tyrosine and threonins - Undergoes
surrounds your nerve cells
phosphorylation which regulate enzyme activity

Physical Properties Of Amino Acids

Roles of Amino Acids in The Body
1. SOLUBILITY - All amino acids are water soluble / Polar amino acids
Phenylalanine:
are more soluble in water
•a precursor for the neurotransmitters tyrosine, dopamine,
2. MELTING POINT - HIGH melting point of > 200 ⁰C
epinephrine and norepinephrine
3. TASTE - Sweet, Bitter, Tasteless
•Plays an integral role in the structure and function of proteins and
enzymes and the production of other amino acids 4. APPEARANCE - White crystalline
Valine: 5. UV ABSORPTION - Aromatic amino acids W,F,Y
•helps stimulate muscle growth and regeneration and is involved in
energy production
Some Reactions of the R Group
Methionine:
Millons – phenolic group
•Important role in metabolism and detoxification.
 Hopkins-Cole – indole ring (-1) + (+1) + (-1) = -1

Xanthoproteic – aromatic ring

Sakaguchi – guanidium group Glu2-

Nitroprusside – sulfhydryl group ✓ Since the pK of NH3+ is 9.7 and the pH increased > 9 therefore the
NH3+ will dissociate → NH2
Sullivans – sulfhydryl group

Pauly – imidazole ring ✓ COO- + NH2 + COO-

(-1) + 0+ (-1) = -2

Test For Amino Acids

Acid- Base Properties
1. POLYPEPTIDE + HOT HCL
•Amino, Carboxylic acid groups readily ionize
→Cut peptide bonds
•Isoelectric Point (pI)
→Release free amino acids
** pH where net charge is 0 **
→Reaction with N- hydroxysuccinimidyl carbamate
•pI = the average of 2 pK’s
→Fluorescent derivative
pI of Basic Amino Acid
→Can be separated and identified
•pI (isoelectric point) = average of 2 pK’s
Nomenclature
•if basic amino acid: average of BASIC groups’ pK
PEPTIDE

→< 50 amino acid

Glutamic Acid Titration Curve
→Low molecular weight
Glu0
OLIGOPEPTIDES
✓ • Since the pK of COOH above is 2.1 and the pH increased > 2
therefore the COOH above will dissociate → COO- →2-10 amino acids

✓ • COO- + NH3+ + COOH →Dipeptide: 2 amino acids

(-1) + (+1)+ 0 =0 →Tripeptides: 3 amino acids

POLYPEPTIDE

pI of Acidic Amino Acid →10-50 amino acid

•pI (isoelectric point) = average of 2 pK’s PROTEIN

•if acidic amino acid: average of ACIDIC groups’ pK →>50 amino acids

→1 or more polypeptide chains

Glutamic Acid Titration Curve

Glu+ 2.NINHYDRIN

✓the pK of COOH above is 2.1 →detects amino acid

✓the pK of NH3+ is 9.7 →a purple product with ALPHA amino acid

→a yellow with imine groups of PROLINE

✓the pK of COOH below is 4.3
PEPTIDES
✓the pH is < 1 therefore nothing will dissociate
✓chain of amino acids
✓COOH + NH3+ + COOH
✓10-50 amino acids
0+ (+1)+ 0 =+1
✓Amino acid units are called Residue
✓Since the pK of COOH below is 4.3 and the pH increased > 4
therefore the COOH will dissociate → COO- Oligopeptides
✓COO- + NH3+ + COO- ✓<10 amino acids
 •Done at successively greater speed, each yielding a pellet and
supernatant.
Peptide bond (AMIDE BOND)
•The faster the speed of the centrifuge, the faster the smaller
•Covalent bond between two amino acids substance will precipitate
•Formed from condensation reaction with liberation of H2O •content of final supernatant corresponds to the cytosol
•Stable and thus, difficult to hydrolyze

•Partial double bond character (10% shorter than standard single

bond)
1.EXTRACTION

Avoids extreme pH and osmotic pressure and high

Evidences Of Peptide Bonds
temperature
1.Biuret test
Employs aqueous solution
general test for proteins
T = 0 – 4 C̊ to avoid loss of biologic activity
(+) if there are at least 2 peptide bonds
2. HOMOGENIZATION
Resonance on the Bond
Disrupts the cell to liberate its constituents, resulting
✓Two amino acid molecules can be covalently joined through a suspension contains intact organelles known as homogenate
substituted amide linkage, termed a peptide bond, to yield a
dipeptide. Such a linkage is formed by removal of the elements of
water (dehydration) from the alpha-carboxyl group of one amino
acid and the alpha-amino group of another
Differential ✓ repeated centrifugation at progressively
Parts of the peptide chain higher speeds
•Amino acids present in peptides are called aminoacyl residues, and centrifugation will fractionate cell homogenates into their
are referred to by replacing the -ate or -ine suffixes of free amino components.
acids with -yl (eg, alanyl, aspartyl, tyrosyl).
✓ used to get the organelles you’re targeting through the
•Peptides are then named as derivatives of the carboxy terminal precipitate becoming smaller and smaller.
aminoacyl residue. For example, Lys-Leu-Tyr-Gln is called lysyl-
leucyl-tyrosyl-glutamine. • the faster the centrifuge, the smaller the pellets.

• at low speed centrifugation the pellet contains whole cells,

nuclei, and cytoskeletons
2.Titratable amino and carboxyl groups
• At high speed, organelles and macromolecules are present
Titratable- FREE amino (NH2 and COOH) groups in the pellet
Amino groups are proton acceptors

3.Hydrolyzed by enzymes specific to certain peptide bonds Experimental Approach Used In Biochemistry
Specific enzymes cut on specific peptide bonds 1.Isolation of biomolecules and organelles

A. Salt fractionation
4.X-ray diffraction studies in hemoglobin & myoglobin B. Chromatography
5.Synthesis of insulin C. Gel filtration

D. Electrophoresis
Experimental Approach Used In Biochemistry E. Ultracentrifugation
SUBCELLULAR ✓ Process of isolating specific organelles
in relatively
2. Determination of structure of biomolecules methods:
FRACTIONATION pure form, free of contamination by
other organelles Elemental analysis

UV, visible, Infrared, NMR spectroscopy

3.CENTRIFUGATION Use of acid/alkaline hydrolysis to degrade the biomolecules to its

basic components
 Use of enzymes

Mass spectrometry analyzes the structure of the separated

substance.

Sequencing methods (for proteins and nucleic acids)

X-ray crystallography