Samenvatting
Samenvatting Biomoleculen (Y)
- Instelling
- Universiteit Leiden (UL)
Samenvatting van het vak Biomoleculen. Zowel de werkgroepen, literatuur, als colleges zijn verwerkt.
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Voorbeeld 4 van de 45 pagina's
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Voorbeeld van de inhoud
Aantekeningen BioMoleculenKleine Moleculen
Chiral molecules Not superimposable on its mirror image
C-Atoms Usually C, bonded to 4 different groups (not atoms! see propyl - methyl)
Isomers
Constitutional/Structural Difference in bonding (same atoms, different molecules)
Stereoisomers Difference in 3Dconfiguration (same atoms, same bonding)
Enantiomers Mirror images of each other
Diastereoisomers Stereo + enantiomers
Meso compounds Molecules with chiral molecules that are mirror-images of e.o.
(superimposable on its mirror image)
Epimers stereoisomers that differ only in configuration around one of
several chiral carbon atoms
Amino acids basic structure → central carbon atom bonded to a carboxyl- +
amino- +
hydrogen- + variable group
Carbohydrates general formula of (CH2O)n with n =/> 3
simplest forms → monosaccharides (sugars, most common is
glucose)
often make up larger polymers + are used for energy storage and
structural components
Nucleotides basic unit of DNA + RNA
molecular currency: ATP (adenine + ribose + 3 phosphates)
5-carbon sugar + nitrogen-containing ring + 1 or more phosphate groups
Lipids Poorly soluble in water (largely composed of long chains of hydrocarbons)
Nucleic acids (see fig. 9.4 Campbell)
Primary structure order of bases in the polynucleotide sequence
Secondary 3D conformation of the backbone (side chain not considered)
Tertiary supercoiling of molecule
The monomer of nucleic acids are nucleotides (nitrogenous base, sugar, phosphoric acid residue)
NA bases a one- or two-ring nitrogenous aromatic compound, two types;
pyrimidines → cytosine, thymine, uracil
purine → adenine, guanine
‘unusual’ bases modified by methylation (mostly in transfer RNA)
Nucleoside base + sugar (glycosidic linkage)
ß-D-ribose vs ß-D-deoxyribose → (deoxy)ribonucleoside
Glycosidic linkage from C-1’ of sugar to N-1 in pyrimidines or N-9 of
purines (sugar atoms primed, N-glycosidic bond)
Ring atoms of the base and the carbon atoms of the sugar (primed) are both numbered.
When phosphoric acid is esterified to one of the hydroxyl groups of the sugar portion of a
nucleoside, a nucleotide is formed. The resulting repeated linkage is a 3’,5’-
phosphodiester bond (3’ → free hydroxyl group, 5’ → phosphate group)
Polymerization of nucleotides gives rise to nucleic acids
Linkage between monomers involves formation of two ester bonds by phosphoric acid. The
hydroxyl groups to which the phosphoric acid is esterified are those bonded to the 3’ and 5’
carbons on adjacent residues (3’,5’-phosphodiester bond)
See for abbreviated structures p 241 (pA → 5’-AMP, Ap → 3’-AMP)
,Bases of nucleic acids (not an alkaline compound, a 1-/2-ring aromatic compound):
Pyrimidine bases: Cytosine, Thymine (primarily DNA), Uracil (RNA) (single-ring)
mostly tRNA → additional bases (see fig. 9.2
Campbell)
Purine bases: Adenine and Guanine (Double-ring)
Carbohydrates
Major energy sources, key role in processes that take place on the surfaces of cells, essential
structural components of several classes of organisms (exampals: glycogen, starch, cellulose)
Monosaccharides formula of Cn(H2O)n
aldose or ketose
6-carbon sugars abundant in nature, 5-structures RNA and DNA,
4+7-photosynthesis + metabolic pathways
formation of oligosaccharides or polysaccharides involves the loss of one
H2O for each new link, accounting for difference in general formula
In a Fischer projection, bonds written vertically on the 2D paper represent bonds directed behind the
paper in three dimensions, whereas bonds written horizontally represent bonds directed in front of the
paper in three dimensions.
The cyclization of sugars takes place as a result of interaction between functional groups
on distant carbons → C-1 + C-5 to form hemiacetal (aldohexoses), C-2 + C-5 to form
hemiketal (ketohexoses). Carbonyl carbon becomes the anomeric carbon (chiral center). Two possible
forms, designated α and ß. This is determined by the hydroxyl group on the carbon that carries the
aldehyde or ketone.
Haworth projection formulas are more useful for representing the overall shape of these kinds of
molecules, shown as planar 5- or 6-membered rings (fura- and pyranose).
D sugar any group that is written to the right of the carbon in a Fischer projection has
a downward direction in a Haworth projection, any group written to the left has
an upward direction. The terminal -CH2OH group (carbon atom with highest
number) is shown in an upward direction. In the α-anomer, the hydroxyl on
the anomeric carbon is on the opposite side of the ring from the terminal -
CH2OH group (pointing down), In the ß-anomer its on the same side (pointing
up).
Glycosides
A glycosidic bond links a sugar and some other moiety.
A sugar hydroxyl group (ROH) can bond to the anomeric carbon to react with another hydroxyl (R’--
OH) to form a glycosidic linkage (R’ -- O -- R). This glycoside is not an ether, glycosides can be
hydrolyzed to the original alcohols. A hemiacetal carbon can react with an alcohol such as methyl
alcohol to give a full acetal, or glycoside. Glycosidic bonds between monosaccharide units are the
basis for the formation of oligo- and polysaccharides. See for notation p 501.
When os and ps form as a result of glycosidic bonding, their chemical natures depend on which
monosaccharides are linked together and on the particular glycosidic bond formed. Both linear and
branched-chain polymers can be formed.
See pp 502/502 → only if the end residue is a free hemiacetal rather than a glycoside will
there be a positive test for a reducing sugar.
Branched homopolysaccharides of glucose
Starches are polymers of α-D-glucose that occur in plant cells. Types of starches can be distinguished
by their degree of cellular branching. See p 509 for examples. Enzymes that hydrolyze starches in
order to release glucose when energy is needed, are α- and ß-amylase (attack α(1 → 4) linkages
,(endoglycosidase (hydrolyzes a glycosidic linkage anywhere along the chain to produce
maltose and glucose)) and exoglycosidase (cleaving nonreducing end of the polymere,
produces maltose)).
Debranching enzymes degrade the α(1 → 6) linkages, in combination with the amylases
degradation of both forms of starch are completed.
Glycosaminoglycans
Glycosaminoglycans are the type of polysaccharide based on a repeating disaccharide in
which one of the sugars is an amino sugar and at least one of them has a negative
charge owing to the presence of a sulfate group or a carboxyl group. Glycosaminoglycan
residue + polypeptide chain → glycoprotein.
Tissue preparation
First step: fixation to preserve structure (most common formalin)
terminate cell metabolism, prevents enzymatic degradation of cells and tissues by
autolysis, kills pathogenic microorganisms and hardens the tissue as a result of cross-
linking or denaturing protein molecules.
Second: preparation for embedding in paraffin to permit sectioning
fixation - washing - dehydrating - clearing - infiltration with melted paraffin - microtome
- mounting medium
Third: staining to permit examination
dissolvement of paraffin - rehydration - staining - dehydration - counterstaining -
mounting medium - covered to obtain permanent preparation
Fixative used for electron microscopy: osmium tetroxide (excellent preservation of membranes)
Autoradiography: Radioactively tagged precursors of molecules are incorporated by cells and
tissues before fixation
Makes use of a photographic emulsion placed over a tissue to localize
radioactive material within tissues
Components remaining after fixation or mostly large molecules that don’t readily dissolve:
- Nucleoproteins
- Intracellular cytoskeletal proteins complexed with associated proteins
- Extracellular proteins bound to molecules by cross-linking
- Membrane phospholipid-protein - / carbohydrate complexes
Lost during routine preparation of H&E-stained sections are small proteins + small nucleic acids, but
also glycogen and proteoglycans and glycosaminoglycans. Also soluble components, ions and
small molecules.
Chemical basis of staining
Acidic dye (eosin): net negative charge on colored portion, general formula [Na +dye- ]
Reacts with cationic groups, particularly with ionized amino groups of proteins →
acidophilia
Neither as specific nor precise as basic dye, but often used in combination or sequentially.
Substances within cells and extracellular matrix that display acidophilia: most cytoplasmic
filaments + most intracellular membranous components + unspecialized cytoplasm +
most extracellular fibers
Basic dye: net positive charge on colored portion, general formula [dye +Cl- ]
Hematoxylin has properties that closely resemble those of a basic dye by usage in combination
with a mordant
, Reacts with anionic (net negative charge) components of cells and tissue
(phosphate groups of nucleic acids, sulfate groups of glycosaminoglycans, carboxyl
groups of proteins) → basophilia
High pH (10) → all three ionized and available for reaction by electrostatic
linkages
Slightly acidic to neutral (5-7) → only sulfate and phosphate
Low pH (below 4) → only sulfate
Substances within cells and extracellular matrix that display basophilia: heterochromatin and
nucleoli of the nucleus + cytoplasmic components + extracellular materials
Cells and tissue structures that have high concentration of ionized sulfate and phosphate groups exhibit
metachromasia.
The periodic acid-Schiff reaction (PAS) stains carbohydrates and carbohydrate-rich molecules. The
basis is formed for the PAS and Feulgen reactions by the bleached basic fuchsin (Schiff reagents)
having the ability to react with aldehyde groups resulting in a distinctive red color. The Feulgen stain
relies on a mild hydrochloric acid hydrolysis to stain DNA.
Stoichiometric reactions: The product of the reaction is measurable and proportional to the
amount of...
Enzyme digestion of a section adjacent to one stained for a specific component can be used to confirm
the identity of the stained material.
Histochemical methods are also used to identify and localize enzymes in cells and tissues.
The reaction product of the enzyme activity is visualized: a capture reagent is used
AB + T 一 (enzyme) → AT + B AB = substrate, T = trapping agent
The specificity of a reaction between an antigen and an antibody is the underlying basis of
immunocytochemistry.
Antibodies (immunoglobulins) are glycoproteins that are produced by specific cells of the
immune system in response to a foreign protein or antigen.
Fluorescent dyes (fluorochromes) are chemicals that absorb light of different wavelengths and
then emit visible light of a specific wavelength. Antibodies can be purified from blood and
conjugated with fluorescein, then applied to sections of lightly fixed or frozen tissues to localize
an antigen in cells and tissues.
Two types of antibodies are used in immunocytochemistry: polyclonal antibodies that are produced by
immunized animals and monoclonal antibodies that are produced by immortalized antibody producing
cell lines.
Both direct (use of one primary antibody) and indirect (considerably enhances the fluorescence signal
emission from the tissue) immunocytochemical methods are used to locate a target antigen in cells and
tissues (see p. 539 Histology).
Determination of the resolution: d= λ / NAobjective + NAcondenser
d = point to point distance of resolved detail in nm
λ = wavelength of light used
NA = numeric aperture or sine of half angle picked up by the objective or condenser of a central
specimen point multiplied by the refractive index of the medium between objective or
condenser and specimen.
Dependent on optical system, wavelength of light source, specimen thickness, quality of
fixation, staining intensity.
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