Académique Documents
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HANDOUT
FOR
BIOCHEMISTRY
Prepared by:
Ser Loisse R. Mortel
with major contributions from Justin Redd B. Mapalo
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An amino acid.
However, we do not consider all amino acids as
important for discussion. In biochemistry, we only
use 20: only those which are essential for the
creation of proteins. They are specifically called alpha
amino acids, because the amino and carboxyl groups
lie on the same (alpha) carbon.
Zwitterionic form - +1 and -1 charge lying on the amino and carboxyl groups, respectively at neutral pH, giving a
net charge of 0. Amino acids always exist with at least one formal charge in the body.
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One letter
Amino acid
abbreviation
P
P henylalanine
V
V aline
T
T hreonine
W
T ryptophan
I
I soleucine
M
M ethionine
H
H istidine
A
A rginine
L
L eucine
K
L ysine
The ten essential amino acids.
The IpH can be computed by getting the average of the two pKa values that flank the 0 net charge. For histidine,
the IpH is 7.585 (IpH = (6.0 + 9.17) / 2).
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Condensation reaction between two amino acids, leading to a dipeptide and water.
N-terminal end of a peptide/protein with an exposed amino group.
C-terminal end of a peptide/protein with an exposed carboxyl group.
Residue R groups of the bonded amino acids in a peptide/protein.
Bonds in peptides
Psi ( ) bond between the alpha carbon and the carboxyl carbon
Phi () bond between the alpha carbon and the amino nitrogen
Omega () - AKA peptide bond
At pH 1, all titratable groups are protonated. This gives the tripeptide a net charge of +1. At pH 1.71, the carboxyl
group loses its protonation giving it a negative charge. This gives the tripeptide a net charge of 0, and the
zwitterionic form is achieved.
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At pH 8.33, the thiol group loses its protonation. This gives the tripeptide a net charge of -1. At pH 9.15, the amino
group is deprotonated. This gives this tripeptide a net charge of -2 from the charged carboxyl and thiol groups.
- The IpH of the tripeptide is 5.02.
B) -pleated Sheet
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D. - complexation interaction of
aromatic molecules with each other
(stacking)
4. QUATERNARY LEVEL OF ORGANIZATION
Same interactions occur but between two
or more polypeptide chains.
Supersecondary Structures
Combinations of -helices and -pleated
sheets (ex. , -hairpin, -meander,
Greek Key, -barrell etc.)
Domains independently folded
structures
Motifs repeated supersecondary
structures
3.
Collagen
Structural protein; filamentous
Left-handed triple helix containing 3.3
residues per turn
Contains 800 residues
Distinctive peptide chains containing a
proline or hydroxyproline residue bound
between a glycine residue and another
amino acid (x pro gly or x hyp gly)
Proline is converted into hyroxyproline by
an enzyme known as hydroxylase which
is catalyzed by Vitamin C.
Synthesis of Collagen:
i. Translation into
preprocollagen.
ii. Hydroxylation: formation of
hydroxyproline and
hyroxylysine
iii. Release from ribosomes
and addition of
endoplasmic reticulum
sugars like galactose and
glucose at hydroxylysine.
iv. Formation of the triple
helix and folding of
globular domains;
transformation into
procollagen
v. Secretion from cell
vi. Removal of N and C
terminal domains;
transformation into
tropocollagen
vii. Deamination of lysine to
form allysine
viii. Cross-linkage of allysine
with a Lysine residue, or
another allysine residue to
form collagen.
2.
Elastin
Hemoglobin
Transport protein; globular
Complex of heme, an iron containing
prosthetic group, and globin, the protein
portion which prevents the oxidation of
Fe 2+ into Fe3+
4.
7.
Myoglobin
Transport protein; globular
Found mainly in the muscles
Composed of only one protein chain with
a heme prosthetic group in the center
Myoglobins oxygen binding curve is
hyperbolic instead of sigmoidal
Immunoglobulins
5.
6.
SOLUBILITY/POLARITY
a. Isoelectric Precipitation adjusting the
pH of the solution until the protein
reaches its IpH and becomes insoluble
b. Salting Out removing water from
proteins by adding an excess amount of a
salt; makes the protein less soluble
(salting in increases the solubility of
proteins in water by adding a sufficient
amount of a salt)
c. Normal and Reversed Phase
Chromatography protein separation
based on affinities with the mobile or
stationary phases; in normal phase
chromatography, the polar proteins are
the last to be eluted
d. High Performance Liquid
Chromatography (HPLC) uses a column
pre-packed with the stationary phase and
is pressurized
2.
MOLECULAR SIZE/WEIGHT
a. Dialysis movement of particles through
a semi-permeable membrane; large MW
proteins will remain inside the dialysis bag
b. Ultrafiltration filtration using a vacuum
c. Ultracentrifugation centrifugating a
solution at various speeds will separate its
Insulin
Regulatory protein/ hormone
Produced from the -cells of the Islets of
Langerhans
Composed of two polypeptide chains,
containing 51 amino acid residues, linked
together
by
intermolecular
and
intramolecular disulfide linkages
Also known as hypoglycemic hormone
Breaks down glucose in a process known
as glycolysis to synthesize pyruvate then
ATP
Converts glucose into glycogen, to be
stored in the liver, through a process
known as glycogenesis
Aids in fatty acid synthesis and protein
synthesis
Glucagon
Also a regulatory protein/hormone
Brings about glycogenolysis
Produced from the -cells of the Islets of
Langerhans
Single polypeptide chain composed of 29
amino acid residues
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d.
e.
3.
CHARGE
a. Electrophoresis separates the proteins
(or amino acids) based on their attraction
towards the negatively charged cathode
or the positively charged anode at a
specific pH
b. Ion Exchange Chromatography uses
cation or anion exchangers (resins); when
a cation exchanger is used, positively
charged amino acids are eluted last
4.
BINDING AFFINITY
a. Affinity Chromatography a ligand acts
as the stationary phase to entrap the
protein of interest
b. Precipitation by Antibodies
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performs
rearrangement
reactions.
6. Ligases performs condensation reactions
along with the expense of energy in the form of
ATP.
Implications:
If the Km is low, it means that it takes just a few
amount of substrate to reach half the Vmax
(thus, the substrate greatly influences enzyme
activity) and vice-versa.
Turnover number - denotes how much
molecules of the substrate turn into product in
a given amount of time. Obviously, higher
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Different types of inhibition in their Lineweaver-Burk plots (both x and y values of Michaelis-Menten plots reciproca ted). Yintercep t is th e recip rocal of Vmax.
=============================================================================================
PART 2: STRUCTURE
Nucleic acids consist building blocks called
nucleotides.
Nucleotides
have
three
components:
1. NITROGENOUS BASE
Pyrimidines: Cytosine, Thymine, Uracil
Purines: Adenine, Guanine
2. FIVE-CARBON SUGAR
RNA: D-Ribose; DNA: 2-Deoxy-D-Ribose
3. PHOSPHATE GROUP
STABILITY ISSUES
Electronegativity disturbances between adjacent
hydroxyl lone pairs may disrupt the stability of
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nucleic
acid
composed
of
deoxynucleotides.
- primary function is storage of genetic
information.
- present in eukaryotic and prokaryotic cells
alike.
- In eukaryotic cells, DNA are complexed with
basic histones.
LEVEL OF ORGANIZATION
1. Primary structure consists of a single
polynucleotide chain of deoxynucleotides.
- Denotes the sequence of nucleotides
(specifically the bases)
2. Secondary structure the more well known
helix structure of DNA, actually is the hydrogen
bonded complex
of two intertwining
deoxynucleotide strands. This is the more
common level of organization in cells.
3. Tertiary structures have been proposed,
usually denoting a circular or intertwining double
helix. A common term associate to this would be
plasmids and topoisomerases.
- Some consider the complex of
deoxyribonucleic acids with histones as the
quaternary level of organization.
PROPERTIES OF THE DOUBLE HELIX
1. Right-handed
2. Antiparallel arranged in opposite
directions
3. Double helix is stabilized by base pair
hydrogen bonds and stacking aromatic
bases (VDW)
Be informed that bases do not hydrogen
bond randomly. They have specificities.
Purine HBs with Pyrimidine
LEVELS OF ORGANIZATION
1. Primary structure - consists of a single
polynucleotide chain of nucleotides.
Denotes the sequence of nucleotides
(specifically the bases)
2. Secondary structure - consists of
interactions within the single strand of an
RNA. This includes hairpin turns, bulges,
and loops.
Base pairs can only happen
intramolecularly, because RNA is always
single stranded.
TYPES OF RNA
RNA has varying designations in the Central
Dogma, unlike DNA. We can apply RNA in the
Central Dogma if we understand the different
types along with the introduction of the
Dogma itself:
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PHASE 1: REPLICATION
DNA -----------> RNA -----------> Protein
PART 1: INITIATION
An origin of replication is recognized
in the DNA.
A replication bubble will then expose
the two separate individual strands to
enzyme action.
PHASE 2: TRANSCRIPTION
the code for translation is understood
before decoding
DNA directed RNA synthesis
is the process by which the DNA serves
its purpose as a storage information
towards production of transfer and
expression material (genes) for the last
phase of the Central Dogma
Consists of four major phases: Initiation,
Elongation, and Termination, and Posttranscription
PART 2: ELONGATION
The elongation starts by the release of
the holoenzyme sub-unit called the
sigma sub-unit. This hints the purpose
of the said sub-unit.
The core enzyme left continues the
whole elongation, using the necessary
nucleotides and Mg++ again to promote
phosphodiester bond formation.
Elongation proceeds in the 5'-3'
direction again.
PART 3: TERMINATION
Termination in transcription, unlike in
replication, can end in two ways:
1. Rho-dependent - using rho-factor to
dissociate the core enzyme from the RNA
sequence being created
PART 4: POST-TRANSCRIPTION
Post-transcription consists of processes that
RNA undergo before being brought out for
their specific purpose. In other words, RNA
has to become mature.
Post-transcription depends on the RNA
being produced because different RNA have
different function, and consequently their
structure. The most tackled is that of
mRNA.
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PHASE 3: TRANSLATION
-
THE GENE
A part of the gene is equivalent to a single
amino acid. This part is a triplet of gene
nucleotides called a codon.
The tRNA which translates the gene reads
the correct codon by a complementary
anticodon.
As more codons are being read, more
amino acids are being connected together
by tRNAs to form the primary structure of
the protein.
The reading of the gene proceeds from
the genes 5-end to its 3-end, and the
translated protein is produced from the N
terminal to the C terminal.
Properties of gene translation:
1. Triplet and nonambiguous rule. A
pattern of three nucleotides code for only
one amino acid.
2. Degenerate. An amino acid may have
more than one codon coding for it.
3. Non-overlapping. Nucleotides for a
codon cannot be read again on the
succeeding codon.
4. No punctuation. The nucleotide next to
the last nucleotide of a codon will
automatically be the first nucleotide of the
next codon.
5. Almost universal. Most species have the
common codon-amino acid correlation.
tRNA
tRNA reads the mRNA sequence while
processing the amino acid sequence.
tRNA further aids translation efficiency
and accuracy by considering wrong
codon sequences thru wobble bases.
THE RIBOSOME
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Shine-Dalgarno
Sequence N-formylmethionine
Eukaryotes AUG - methionine
3. The 50S subunit is binded with tRNA by
the guidance of IF2. The resulting complex
is called the 50S initiation complex.
4. The initiation complexes combine. The
resulting complex is called the 70S initiation
complex.
5. The tRNA for the first codon goes to the
P region and hydrogen bonds using the
respective anticodon
Usage of GTP
PART 2: ELONGATION
1. Elongation factor detects functional
codon
The tRNA for the first codon moves
toward the A region thru the elongation
factor (uses GTP)
2. A peptide bond between the first and
second amino acids held by the first and
second tRNAs is catalyzed by peptidyl
transferase; tRNA on P (the first one) is cut
from its amino acid
At this point, the second tRNA on the A
site holds both amino acids
3. Translocation of second tRNA from A to
P, where the first tRNA goes from P to E to
exit (uses GTP)
4. Arrival of new tRNA on A by elongation
factors and process repeats for many times
PART 2: INITIATION
1. The ribosomal subunits are separated
together by Initiation Factor (IF) 1.
2. mRNA combine with the lighter subunit
and place the first two codons to the P and
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PART 3: TERMINATION
1. Release factors recognize stop codons
2. A release factor goes to the A region
3. The ester bond between the P tRNA and
attached amino acid is cut off.
Synthesis stops and the initiation
complexes disassemble.
Modification
The primary structure itself may be
enzymatically modified to its native
form by the following ways:
1. The N-terminal amino acid is
trimmed away
2. Amino acid residues are
modified to become other
residues
(ex. Hydroxylation)
The native protein may be modified to
its functional form by addition of
prosthetic groups or bonding with
other biomolecules.
Degradation
Mutations in the gene may go through
the proofreading mechanism of tRNA
and create wrong sequences in
proteins.
Wrong proteins can be degraded by
proteases/proteasomes.
Degradation is a well-controlled
process. They usually need molecular
signals that trigger these enzymes
Ubiquitin-proteasome pathway
common pathway using ubiquitin as a
signal for protease activation towards
the protein it is binded to
Ubiquitin is a 76 amino acid
polypeptide
Ubiquitin binds with a very positively
charged N-terminal of a non-functional
protein by help of E3 (ubiquitin ligase)
E1 ubiquitin-activating enzyme
E2 ubiquitin-conjugating enzyme
Acidic N-terminal residues addition
of arginine by Arg-tRNA, consequently
attracting ubiquitin attack through
ubiquitin ligase
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Protein Targeting
Proteins that are mature and fully
functional must be delivered to the
right parts of the cell.
Proteins may be delivered toward the
cytoplasm or through membranebound organelles.
Amino acid sequences within the
protein (signal sequences) trigger
transfer by the proper organelles.
Endoplasmic reticulum site where
ribosomes produce proteins that have
to go through membranes, lysosomes
or export.
- further modification of protein by removal
of nonfunctional signal sequences or
attachment of other biomolecules
2. Chemical
a. Superoxide species apyrimidinic site
b. Alkoxy free radicals - dimerization
c. Amino acid pyrolysates adduct with
guanine
d. Nitrosoamines apurinic site/misreading
e. Polycyclic aromatic hydrocarbons
disrupts the DNA helix
f. Hydrazines creates rigid loop
g. Caffeine may disrupt repair system
h. Bisulfite (HSO-) can cause misreading of
C as U
j. Dibromoethylene dipositivity
k. Aflatoxin
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a)
Glucose, an aldohexdose.
b)
Table of a) aldoses and b) ketoses, respectively.
Another essential thing to note In discussing carbohydrates is that we use two structural formulas to draw them.
The first one also used in the aldoses/ketoses chart above is called the Fischer Projection Formula, which is used
for the linear form of carbohydrates.
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Conformational isomers of carbohydrates are simply shape-based isomers of the exactly same cyclic
carbohydrate. The envelope configuration exists for pentoses, while the chair and boat configurations exist for
hexoses.
Alduronic acid
Aldaric acid
A carboxylic acid product of sugar oxidation can cyclicize, this time, doing esterification by S NAcyl (same concept as
hemiacetal formation, but this time with carboxyl instead of keto or formyl group). The product cyclic ester is also
called a lactone. However, the requirement for oxidation of cyclics is the presence of a free anomeric carbon.
2. Reduction polyhydroxy alcohols/alditol if carbonyl is reduced (ex. Glucose to glucitol(sorbitol) below)
- deoxy sugars if hydroxyl is reduced/removed (ex. Deoxyribose below, where C2 OH is reduced to H)
3. Acetal/Ketal Formation reacting a hydroxyl compound with a cyclic sugar (hemiacetal/hemiketal), thus
forming an acetal/ketal which is now called a glycoside.
Glycosides compounds wherein there is addition of an alcohol to a free anomeric carbon; an acetal/ketal
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Glucose
Energy investment
Energy payoff
G3P
Pyruvate
ATP
Decarboxylation of Pyruvate
Electron carrier
ATPs produced
2.5 (or 3)
FADH 2
1.5 (or 2)
GTP
Energy investment phase Glucose is broken into two molecules of G3P (Glyceraldehyde-3-phosphate).
It consists of the first 5 steps of glycolysis.
Energy payoff phase G3P molecules are turned into pyruvate. It consists of the last 5 steps of
glycolysis.
Enzyme
1, phosphorylation
2, isomerization
3, phosphorylation
4, cleavage and oxidation
Hexokinase
Phosphoglucose isomerase
Phosphofructokinase
Aldolase
5, isomerization
Product
Glucose-6-phosphate
Fructose-6-phosphate
Fructose-1,6-bisphosphate
Glyceraldehyde-3-phosphate
(G3P);
Dihydroxyacetone
Glyceraldehyde-3-phosphate
Energy carrier
involved
- 1 ATP
-1 ATP
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6, oxidative phosphorylation
7, substrate-level
phosphorylation (of ATP)
8, isomerization
9, dehydration
10, substrate-level
phosphorylation (of ATP)
G3P dehydrogenase
Phosphoglycerate kinase
1,3-bisphosphoglycerate
3-phosphoglycerate
+ 1 NADH
+ 1 ATP
Phosphoglycerate mutase
Enolase
Pyruvate kinase
2-phosphoglycerate
Phosphoenolpyruvate (PEP)
Pyruvate
+ 1 ATP
A critical stage after glycolysis is the transfer of NADH from the cytoplasm into the electron transport chain to
produce the promised number of ATPs from it. With its size, NADH must be supported by a shuttle to enter the
mitochondrial membrane, where ETC is located. Two shuttles exist: Malate-aspartate (MA) shuttle, and Glycerolphosphate (GP) shuttle. MA Shuttle requires no energy, while GP shuttle requires one mole of ATP per mole of
NADH. Thus, variations in the total number of ATP after the ETC result from this shuttle.
Glycogen enters a slightly different catabolic pathway (glycogenolysis), being catabolized into glucose-1phosphate by phosphorylase enzyme, then isomerized into glucose-6-phosphate, both without using energy. G3P
then enters the usual glycolytic pathway. The ATP consumption for phosphorylation of glucose to glucose-6phosphate (step 1) is then conserved.
Hexokinase targets hexoses in general, not just glucose, and thus will produce the same energy output with
hexoses other than glucose.
After glycolysis, pyruvic acid enters two pathways: an oxygen-including Tricarboxylic Acid Cycle or anaerobic
respiration. Obligate anaerobes and facultative aerobes engage in a form of respiration that is much unlike that
of aerobic respiration. In addition, anaerobic respiration usually discussed in books are very simple compared to
their aerobic counterpart. They always start with glycolysis to produce the pyruvic acid, and continue to the
following steps.
Pyruvate decarboxylase
Pyruvate
Alcohol dehydrogenase
Acetaldehyde
Ethyl Alcohol
NADH NAD
The Ethanol fermentation process utilizes Pyruvate, first decarboxylating it to form Ethanal (Acetaldehyde),
then finally reducing it through NADH to produce Ethyl Alcohol.
Pyruvate
Lactic Acid
NADH NAD
The Lactate fermentation directly reduces pyruvate through NADH to produce Lactate or Lactic acid .
It must be noted that fermentation happens only in the absence of oxygen. In its presence, pyruvate proceeds to
the Tricarboxylic acid cycle.
PDH Complex
Pyruvate
acetyl-CoA
NAD -> NADH
Enzyme
Product
Energy carrier
involved
Pyruvate dehydrogenase
(PDH) complex
Citrate synthase
Aconitase
Acetyl-CoA
Isocitrate decarboxylase
-ketoglutarate
Succinyl-CoA
+1 NADH
+1 NADH
5, substrate-level
phosphorylation (of ATP)
6, dehydrogenation (oxidation)
7, dehydrogenation
8, oxidative dehydrogenation
Succinate thiokinase
Succinate
+1 GTP
Succinate dehydrogenase
Fumarase
Malate dehydrogenase
Fumarate
Malate
Oxaloacetate
+1 FADH2
-ketoglutarate
dehydrogenase complex
+1 NADH
Citric acid
Cis-aconitate -> Isocitrate
+1 NADH
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Pre--oxidation cycle:
O
Note: RCSCoA = fatty acyl-CoA
Enzyme
Fatty acyl-CoA synthetase, Carnitine
acyltransferases I and II
Acyl-CoA dehydrogenase
Enoyl-CoA hydratase
L-3 hydroxyacyl-CoA dehydrogenase
3-ketoacyl-CoA thiolase
Product
Energy carrier
involved
Acyl-CoA
- 2 ATP
Enoyl-CoA
L-3 hydroxyacyl-CoA
3-ketoacyl-CoA
Acetyl-CoA and Acyl-CoA 2
carbons shorter than
original
+1 FADH2
+1 NADH
The special factor in the catabolism of fatty acids is that for a single acid, the cycle presented will repeat several
times, until finally all of the carbon chain is turned into acetyl-CoA. Thus, the cycle will equate to the number of
acetyl-CoA products minus one.
Although the fatty acid metabolism produces a much larger amount of acetyl-CoA that can translate to a large bulk
of ATP, the fact that the TCA cycle is non-simultaneous may just promote unproductive build-up of unused acetylCoA. Moreover, acetyl-CoA is a precursor for cholesterol and ketone bodies, both producing unfavorable effects
on the body.
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Pathway,
reaction #
G1
G3
G6
G7
G10
PTCA
TCA3
TCA4
TCA5
TCA6
TCA8
Product
Glucose-6-phosphate
Fructose-1,6-bisphosphate
1,3-bisphosphoglycerate
3-phosphoglycerate
Pyruvic acid (end of glycolysis)
Acetyl-CoA
Alpha-Ketoglutarate
Succinyl-CoA
Succinate
Fumarate
Oxaloacetate (end of TCA)
PBOC
OC1
Acyl-CoA
Enoyl-CoA
OC2
1, -hydroxyacyl
dehydrogenase
+ 2.5 multiplied by
#C/2 (- 2)
#C = carbons in acid
1.5 x #cycles
+ (10 x number of acetyl
CoA produced)
- 2 (for pre-O cycle)
4 x #cycles
+ (10 x number of acetyl
CoA produced)
- 2 (for pre-O cycle)
NOTE: The double ATP column was meant to reflect the amount of ATP produced per step for EVERY hexose (since
each hexose is split into TWO after the first half of glycolysis).
References:
1. Campbell, M., Farell, S. (2012). Biochemistry 7th Edition. Belmont, CA: Thomson Brooks/Cole.
2. Mauseth, J.D. (2009). Botany: An Introduction to Plant Biology Fourth Edition. Sudbury, MA: Jones and
Bartlett Publishers, Inc.
3. Nelson, D., Cox, M. (2008). Lehninger principles of Biochemistry Fifth Edition. New York: W.H. Freeman and
Company.
4. Boyer, R. (2006). Concepts in Biochemistry 3rd Edition. New York: John Wiley & Sons.
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