WELCOME ON THE BIOCHEMISTRY LECTURES
description
Transcript of WELCOME ON THE BIOCHEMISTRY LECTURES
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2nd semester
An Introduction to Biochemistry
2nd semester
An Introduction to Biochemistry
Bioorganic Chemistry • 1. Amino acids, Protein and Enzymes• 2. Other biomolecules:
• Carbohydrates• Lipids• Nucleotides and Nucleic Acids• Vitamins and Coenzymes
Biochemistry - Bioenergetics • 1. High energy phosphate compounds• 2. Metabolic Pathways:
• Glycolysis• Citrate cycle• Mitochondrial respiration
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Books and NotesBooks and Notes
Lehninger: Biochemistry (selected chapters)
Calculation book (previous semester)Practice book (previous semester)
Collected STUCTURES
You can download the lecture material from our website
www.biokemia.sote.huUsername: file
Password: open2
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2 Midterms during the semester
Assay questions (topics), calculations, structures
Chemistry Final
2nd semester: 30 multiplichoice questions1st semester: 20 multiplichoice questions10 structures
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Amino AcidsThe Building Blocks of Proteins
1. Neutral amino acids(one amino- and one carboxyl group)
Amino AcidsThe Building Blocks of Proteins
1. Neutral amino acids(one amino- and one carboxyl group)
Biochemistry LecturesBiochemistry LecturesAmino Acids, Proteins, EnzymesAmino Acids, Proteins, Enzymes
DR. M. Sasvári
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Amino AcidsAmino Acids
R
C
COO-
+H3N H
-ketoacid derivatives of amino acids
R
C
COO-
O
Grouping of amino acids is based on the side chains:Nonpolar (hydrophobic) – aliphatic and aromatic side chains (only C,H)Polar, uncharged (hydrophylic) – other atoms (O,N,S) than C and HPolar, charged (ionic) – weak acids or bases
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CH3
C
COO-
O
pyruvate
Transamination:-ketoacid
red
ox
Main metabolic intermediers:
Nonpolar (hydrophobic), aliphatic side chainsNonpolar (hydrophobic), aliphatic side chains
CH3
L-Alanine (3C)Ala, A
C
COO-
+H3N H
CH3
C
COO-
HO H
lactate
Reduction:-hydroxy-acid
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Transaminated forms:
CH
CH2
CH3
CH3
Nonpolar (hydrophobic), aliphatic side chainsBRANCHED CHAIN AMINO ACIDS
Nonpolar (hydrophobic), aliphatic side chainsBRANCHED CHAIN AMINO ACIDS
isovalerateketo isocapronateketo isocapronateketo
CH
CH3 CH3
CH2
CH
CH3 CH3
Val, VValine
Leu, LLeucine
Ile, IIsoleucine
C
COO-
+H3N H C
COO-
+H3N H C
COO-
+H3N H
Metabolic disorder in the catabolism of branched chain amino acids:-hydroxy-isovalerate and -hydroxy-isocapronate in the urine
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IleLeuVal
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Neutral Amino Acids: Acid-base character
Neutral Amino Acids: Acid-base character
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Protonated forms Deprotonated forms
-H
HA A- + H+
-carboxyl group is an ACID:
H+
B+ B + H+
-amino group is a base:
pKa2
pH < pKa pKa < pH
pKa1
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Neutral Amino Acids: Protonic equilibria
Neutral Amino Acids: Protonic equilibria
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Form 3. Fullydeprotonated form
Form 2. Isoelectric form
Form I. Fully protonated form
pH < pKa1HH+
H+-
pH > pKa2
pH =(pKa1+ pKa2)/2
pKa1= 2.3
pKa2 = 9.6
H
C COOH+H3N
R
H
C COO-+H3N
R
H
C COO-H2N
R
-
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H
C COO-H2N
R
H
C COO-+H3N
R
H
C COOH+H3N
R
pKa1= 2.3
pKa2 = 9.6
BUFFERS
pH around pKa1
Form 2./ Form 1.BUFFER
pH around pKa1
Form 2./ Form 1.BUFFER
pH around pKa2
Form 3./ Form 2.BUFFER
pH around pKa2
Form 3./ Form 2.BUFFER
NO Buffer at Isoelectric pH!
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Neutral Amino Acids: pH dependence
of different forms
Neutral Amino Acids: pH dependence
of different forms
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2 4 6 8 10 12 pH
5
10
mmol
Different forms of AlaDifferent forms of Ala
+
H
C COOH+H3N
R
+ -
H
C COO-+H3N
R
-
H
C COO-H2N
R
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Buffer 2.Buffer 1.meqv NaOH added
0
2
4
6
8
10
12
14
0 2 4 6 8 10 12 14 16 18 20 21
pH
1st Eqv.Point
Ip
2nd Eqv.Point
Neutral amino acids: Titration curveNeutral amino acids: Titration curve
START:10 mmolcompletely protonated neutral amino acid
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pH = 2.3 + lg (deprot./prot.)
pH = 2.3 + lg 4/6 = 2.1
10 ml of 0.1 M completely protonated glycine solution + x ml 0.1 N NaOH, pH=2.6
pH = 2.3 + lg x/(10-x) = 2.6x= 6.66 ml
See also: Selected Calc.CHAPTER 8. AMINO ACIDS AS BUFFER
10 ml of 0.1 M glycine solution (completely protonated)+ 4 ml 0.1 N NaOH, pH=?
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Aliphatic secondary amine
Gly, GGlycine
Pro, PProline
H
COO-
+H2N
maximal flexibility rigidNo side chain Alicyclic side chain (nonpolar)
Glycine and Proline: Two extremitiesGlycine and Proline: Two extremities
C
COO-
+H3N H
Aliphatic primary amine
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Gly Pro
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AROMATIC AMINO ACIDS hydrophobic interactions between stacking aromatic rings
AROMATIC AMINO ACIDS hydrophobic interactions between stacking aromatic rings
CH2
C
COO-
+H3N H
nonpolar
Phe, FPhenylalanine
phenyl-pyruvate p-hydroxyphenyl-pyruvateTransaminated forms:
Phenylketonuria: phenyl-pyruvate, phenyl-lactate and phenyl-acetate in the urine
Tyr, YTyrosine
Phenolic -OH (very weak acid)
C
COO-
+H3N H
OH
CH2
polar
Phe hydroxylase(deficiency: phenylketonuria)
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AROMATIC AMINO ACIDShydrophobic interactions between stacking aromatic rings
AROMATIC AMINO ACIDShydrophobic interactions between stacking aromatic rings
Trp, WTryptophan
NH
CH2
C
COO-
+H3N H
polar
Aromatic secondary aminein the indol ringVery weak base
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Aromatic amino acids have a characteristic absorbance
at 280 nm
Characteristic absorbance of proteins.
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His, HHistidine
NH+HN
CH2
aromatic imine(weak base)
in the imidasol ring
C
COO-
+H3N H
AROMATIC AMINO ACIDSwith positively charged side chain
AROMATIC AMINO ACIDSwith positively charged side chain
NHHN
CH2
+
delocalized structure
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PheIle Tyr
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Amino Acids: Optical ActivityAmino Acids: Optical Activity
Assymetrical (chiral) C atom (4 different substituents)Enantiomer pairs (mirror images, not imposable on each other)
Optical activity
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D-Glyceraldehyde L-Glyceraldehyde
C OH
CH2OH
CHO
H C H
CH2OH
CHO
OH
Amino Acids: Optical ActivityProteins Consist of L-Amino Acids
Amino Acids: Optical ActivityProteins Consist of L-Amino Acids
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D-Alanine L-Alanine
CH3
C NH2
COO-
H C H
COO-
H2N
CH3
Amino Acids: Optical ActivityProteins Consist of L-Amino Acids
Amino Acids: Optical ActivityProteins Consist of L-Amino Acids
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L- Alanine D- Alanine
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C H
CH3
COO-
OH
CH NH3+
Amino Acids: Optical activitySome amino acids have 2 chiral centers
Amino Acids: Optical activitySome amino acids have 2 chiral centers
D-allothreonineL-allothreonine
L-Thr D-Thr
Enantiomer pairs
Diastereom
ersD
iast
ereo
mer
s
CH
CH3
COO-
OH
C H+H3N
COO-
COH
CH3
H
CH NH3+
COH
CH3
COO-
H
C H+H3N
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Amino Acids: Polar side chains-OH containing side chains
Amino Acids: Polar side chains-OH containing side chains
Thr, TThreonine
Ser, S Serine
CH2 OH CH
CH3
OH
Primary alcohol Secondary alcohol
C
COO-
+H3N H C
COO-
+H3N H
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Phosphate group of ATP
+
O
R1 P OH
OH
Biochemical reaction:
An ALCOHOL reacting with CARBOXYLIC ACID forms ESTERS
Reactions of the Ser OH groupReactions of the Ser OH group
R2 OH+
Alcohol Ester
C
O
R1 O R2 + H2OH+
heat
O
R1 C OH
Carboxylic acid
Chemical reaction:
R2 OH
Ser –OHof a protein
enzyme
Phosphate esteron a protein
P
O
R1 O R2
OH
+ H2O
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Thr
CH
CH3
OH
C
COO-
+H3N H
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CH2 SH
Amino Acids: Grouping principles-S containing side chains
Amino Acids: Grouping principles-S containing side chains
Cys, CCysteine
Met, MMethionine
S: low electronnegativity slight polarity
CH2
S
CH3
CH2
ThioalcoholThioether
C
COO-
+H3N H C
COO-
+H3N H
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Two cysteine units form a disulfide bridge
Reactions of Amino AcidsReactions of Amino Acids
Formation of disulfide bridge
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A schematic diagram of two disulfides from the protein structure of bovine insulin
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Role of Methionin in the metabolism Its activated form is a methyl group donor
Role of Methionin in the metabolism Its activated form is a methyl group donor
CH2
S
CH3
CH2
C
COO-
+H3N H
Methionin (5C)
CH2
SH
CH2
C
COO-
+H3N H
Homocystein (4C)
SH
CH2
C
COO-
+H3N H
Cystein (3C)
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Exchange of the sulfur in the metabolism Exchange of the sulfur in the metabolism
CH2 S
CH2
C
COO-
+H3N H
CH2
C
COO-
+H3N H Cystathionine
Cystein (3C and -SH)Homoserine (4C and -OH)
Homocystein (4C and -SH) Serine (3C and -OH)
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Amino Acids: Polar side chainsNegatively charged (ACIDIC) side chains.
Amino Acids: Polar side chainsNegatively charged (ACIDIC) side chains.
Glu, EGlutamate
Asp, DAspartate
CH2
COO -
CH2
CH2
COO -
-carboxyl -carboxyl
C
COO-
+H3N HC
COO-
+H3N H
Transamination:oxaloacetate
-keto-glutarate
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Amino Acids: Polar side chainsAmides
Amino Acids: Polar side chainsAmides
CH2
CONH2
CH2
CH2
CONH2
amide amide
Amide of Aspartate Amide of Glumatate
Asn, NAsparagine
Gln, QGlutamine
C
COO-
+H3N H C
COO-
+H3N H
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Glu Gln
CH2
COO -
CH2
C
COO-
+H3N H
CH2
CONH2
CH2
C
COO-
+H3N H
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Side chain
NH
Side chain
C O -
CH2
O
Side chain
CH2
C NH2
O
CH2
C NH2
O
Side chain
Hydrogen bonds between side chainsHydrogen bonds between side chains
OH
Side chain
Hydrogen acceptors: Asp, Glu, Asn, Gln
Hydrogen donors: Trp, Ser,Thr,Tyr,Asn, Gln, His
Side chainCH2
C
O
NHH
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Glu
Tyr
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Gln
Tyr
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Gln
Asn
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Biogen aminesBiogen amines
Biochemical reaction: Decarboxylation of amino acids
R
C
COO-
+H3N H
Amino acid
R
C +H3N H2
Biogen amine
CO2
decarboxylation
specificdecarboxylases
(enzymes)
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Biogen aminesBiogen amines
Synthesis of dopamine from Tyr
C
COO-
+H3N H
OH
CH2
C
COO-
+H3N H
OH
CH2
Tyrosine
C
COO-
+H3N H
OH
CH2
C
COO-
+H3N H
OH
CH2
Dopa
OH
Dopamine: neurotransmitterParkinson’s disease - low dopamine production
CO2
decarboxylation
Tyr hydroxylase Aromatic amino acid decarboxylase
hydroxylation
O2 H2O
Dopamine
OH
C+H3N
OH
CH2
C+H3N H2
OH
CH2
Biogen amine
OH
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Neurotransmitter Stress hormone
Biogen aminesBiogen amines
Synthesis of norepinephrine and epinephrine
Dopamine hydroxylase
vitamin C
methylation
methyl group donor
phenylethanol amineN-methyl transferase
hydroxylation
O2 H2O
dopamine
OH
C+H3N
OH
CH2
C+H3N H2
OH
CH2
OH
Norepinephrine(noradrenaline)Biogen amine
OH
C+H3N
OH
CH
C+H3N H2
OH
CHHO
OHOH
C+H N
OH
CH
C+H N H2
OH
CHHO
CH3
2
Epinephrine(adrenaline)Biogen amine
OH
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Biogen aminesBiogen amines
+H3N-C-COO-
CH2
CH2
COO-
H+H3N-C-COO-
CH2
CH2
COO-
H
Glu
+H3N-CH2
CH2
CH2
COO-
+H3N-CH2
CH2
CH2
COO-Gamma-amino butirate
(GABA)biogen amine
neurotransmitter
CO2
decarboxylation
Synthesis of GABA from Glu
Glu decarboxylase
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Biogen aminesBiogen amines
CO2
decarboxylation
His
NHN
CH2
H
C COO-+NH3
NHN
CH2
H
C -+NH3
VasodilatorReleased in allergic response
Stimulation of acid secretion in the stomach
NHN
CH2
H
C+NH3
NHN
CH2
H
C+NH3 H
histaminebiogen amine
Synthesis of histamine from His
His decarboxylase
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Biogen aminesBiogen amines
CO2
decarboxylation
Neurotransmitter (brain)Blood coagulation (trombocytes)
Trp
CH
C
COO -
+H 3N H
CH2
C
COO -
+H 3N H
CH
C
COO -
+H 3N H
CH2
C
COO -
+H 3N H
NH
CH
C
COO -
+H 3N H
CH2
C
COO -
+H 3N H
CH
C
COO -
+H 3N H
CH2
C
COO -
+H 3N H
NH
5-hydroxy-Trp
CH
C
COO -
+H 3N H
CH2
C
COO -
+H 3N H
OHCH
C
COO -
+H 3N H
CH2
C
COO -
+H 3N H
NH
CH
C
COO -
+H 3N H
CH2
C
COO -
+H 3N H
OHCH
C
COO -
+H 3N H
CH2
C
COO -
+H 3N H
NH
Serotoninbiogen amine
CH
C+H 3N
CH2
C+H 3N
OHCH
C+H 3N
CH2
C+H 3N H2
NH
CH
C+H 3N
CH2
C+H 3N
OHCH
C+H 3N
CH2
C+H 3N H2
NH
Synthesis of serotonin from Trp
hydroxylation
O2 H2O
Trp hydroxylase
Aromatic amino acid decarboxylase
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H
C COO-+H3N
CH2
OH
Tyr
thiol alcohol
carboxyl
H
C COO-+H3N
CH2
COO-
Asp
carboxyl
Glu
H
C COO-+H3NCH
2
CH2
COO-
carboxyl
phenol
phenolthiol alcohol
Acidic Amino AcidsComparison of acidity of different groups
Acidic Amino AcidsComparison of acidity of different groups
Acidic amino acids(negative charge at pH 7)
Side chains capable for proton dissociation(no charge on side chain at pH 7)
pKa values in different groups:
2 4 7 10 14
CH2
H
C COO-+H3N
SH
CysH
C COO-+H3N
CH22
OH
Ser
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2nd BUFFER1st BUFFER
Acidic amino acids: Titration curveAcidic amino acids: Titration curve
Titration of 10 ml 1 M Glu (10 mmol) solution with 1 M NaOH:
Glu : 10ml x 1 M = 10 mmol NaOH: x ml * 1 M = x meqv
Ip
3rd BUFFER
0
2
4
6
8
10
12
14
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 31
1st Eqv.Point 2nd Eqv.Point 3rd Eqv.Point
pH
NaOH ml/meqv added
Acidic amino acidshave acidic Ip!
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NaOH Form 1. Form 2. Form 3. Form 4.added Calculation of pH pHmeqv mmol mmol mmol mmol
0 10 02 8 2 Buffer 1. 1.64 6 4 2.06 4 6 pH = 2.2 + lg(Form2./Form1.) 2.48 2 8 2.8
10 0 10 0 Ip: pH = (pKa1+pKa2)/2 3.2
12 8 2 Buffer 2. 3.614 6 4 4.216 4 6 pH = 4.3 + lg(Form3./Form2.) 4.618 2 8 4.9
20 0 10 0 pH = (pKa2+pKa3)/2 7.0
22 8 2 Buffer 3. 9.124 6 4 9.526 4 6 pH = 9.7 + lg(Form4./Form3.) 9.828 2 8 10.330 0 10
Acidic amino acids: Calculations Acidic amino acids: Calculations
Titration of 10 ml 1 M Glu solution with 1 M NaOH:
Glu : 10ml x 1 M = 10 mmol NaOH: (x) ml x 1 M = (x) meqv
5
START:Form 1. - COOH - COOH - NH3
+
Form 2. - COO -
- COOH - NH3
+ Ip
Form 3.
- COO -
- COO -
- NH3+
Form 4.
- COO -
- COO -
- NH2
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Amino Acids: Grouping principlesPositively charged (BASIC) side chains.
Amino Acids: Grouping principlesPositively charged (BASIC) side chains.
His, HHistidine
Lys, KLysine
Arg, R Arginine
CH2
NH2+
CH2
CH2
NH
C
NH2
NH+HN
CH2
CH2
CH2
CH2
CH2
NH3+
C
COO-
+H3N H C
COO-
+H3N H C
COO-
+H3N H
Guanidinogroup
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Delocalization of electrons in Arg and His side chains
Arg: guanidino group
NHHN
CH2
+
His: imidasol ring
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Arg Lys
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His
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pKa values of different groups:
7 10 1412
Basic amino acids: Comparison of basicity.Basic amino acids: Comparison of basicity.
-amino
alkyl imine
imidasol -amino
guanidinoalkyl imine
- aminoalkyl amine
imidasol ringaryl imine
His
NHN
CH2
H
C COO-+NH3
Lys
CH2
CH2
CH2
CH2
NH3+
H
C COO- NH2
Arg
NH
NH
CH2
CH2
CH2
NH
C 2+
H
C COO-NH2
NH
NH
CH2
CH2
CH2
NH
C 2+
H
C COO-NH2
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Isoelectric forms of basic amino acidsIsoelectric forms of basic amino acids
NHN
CH2
H
C COO-+NH3
His Ip: No charge at the side chain
Lys
CH2
CH2
CH2
CH2
NH3+
H
C COO- NH2
ArgNH
NH
CH2
CH2
CH2
NH
C2+
H
C COO-NH2
Ip: Positively charged side chains
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3rd BUFFER
Basic amino acids: Titration curveBasic amino acids: Titration curve
Titration of 10 ml 1 M Arg (10 mmol) solution with 1 M NaOH:
Arg : 10ml x 1 M = 10 meqv NaOH: (x) ml x 1 M = (x) meqv
2nd BUFFER1st BUFFER
0
2
4
6
8
10
12
14
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
1st Eqv.Point 2nd Eqv.Point 3rd Eqv.Point
pH
NaOH ml/meqv added
Ip Basic amino acidshave basic Ip!
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Basic amino acids: CalculationsBasic amino acids: Calculations
Titration of 10 ml 1 M Arg solution with 1 M NaOH:
Arg : 10ml x 1 M = 10 mmol NaOH: x ml x 1 M = x meqv
NaOH Form 1. Form 2. Form 3. Form 4.added Calculation of pH pHmeqv mmol mmol mmol mmol
0 10 02 8 2 Buffer 1. 1.64 6 4 2.06 4 6 pH = 2.2 + lg(Form2./Form1.) 2.48 2 8 2.8
10 0 10 0 pH = (pKa1+pKa2)/2 5.6
12 8 2 Buffer 2. 8.414 6 4 8.916 4 6 pH = 9.0 + lg(Form3./Form2.) 9.318 2 8 9.6
20 0 10 0 Ip: pH = (pKa2+pKa3)/2 10.8
22 8 2 Buffer 3. 11.924 6 4 12.326 4 6 pH = 12.5 + lg(Form4./Form3.) 12.728 2 8 13.130 0 10
Ip
START:Form 1. - COOH - NH3
+
NH2+
Form 2. - COO -
- NH3+
NH2+
Form 3.
- COO -
- NH2
NH2+
Form 4.
- COO -
- NH2
NH
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Reactions of Amino AcidsReactions of Amino Acids
ACYLATION of AMINES forms AMIDES
R2NH2
base
O
R1 C NH – R2
Amide
+ HCl
O
R1 C Cl
Acyl chloride
Chemical reaction:
Biochemical reaction:O
R C OH
Carboxylic acide.g. glutamate
O
R C NH2
Amidee.g. glutamine
+ H2OATP (energy)enzyme
NH3
or amino group donor
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Glu -ketoglutarate
CH2
COO -
CH2
C
COO-
+H3N H
CH2
COO -
CH2
CH2
COO -
CH2
C
COO-
+H3N H
CH2
COO -
CH2
C
COO-
O
CH2
COO -
CH2
CH2
COO -
CH2
C
COO-
OO
AspoxaloacetateCH2
COO -
C
COO-
O
CH2
COO -
CH2
COO -
C
COO-
OO
CH2
COO -
C
COO-
+H3N H
CH2
COO -
CH2
COO -
C
COO-
+H3N H
Biochemical reaction: TransaminationAn exchange of amino and oxo groups
Reactions of Amino AcidsReactions of Amino Acids
Aspartate amino transferase (ASAT)
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A color reaction for amino acids: ninhydrin test
Reactions of Amino AcidsReactions of Amino Acids
Chemical reaction (amino acid will be broken):
Usage: staining of amino acids and proteins (see: practice)
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Reactions of Amino AcidsReactions of Amino Acids
Chemical reaction (amino acid will NOT be broken):
A color reaction for amino acids: Sanger’s reagent
NH2CHOOCH
R
2,4-dinitrofluorobenzene (DNFB)
F
NO2
NO2+
Usage: staining of N-terminal amino acid of proteins (protein sequencing)
NH
NO2
NO2CHOOCH
R
2,4-dinitrophenylamino acid(DNP-derivative)
+HF
DNP will be attached to amino groups in a protein or peptide
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Peptides: N terminal and C terminalPeptides: N terminal and C terminal
N-terminal C-terminal
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Fluorescence labeling of amino acids/proteins
Reactions of Amino AcidsReactions of Amino Acids
Fluorescence - "glow in the dark".
Usage of fluorescamine: - sensitive quantification of proteins- sensitive detection of proteins
Chemical reaction
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Tubulin in a live bovine pulmonary artery endothelial cell labeled with green fluorescent reagent
endoplasmic reticulum was visualized with red-fluorescent reagent