L1&2
1. Proteins:Determination of Primary Structure
proteins perform multiple critically important roles. proteins &
peptides must be purified prior to analysis: column chromatography,
partition chromatography, size exclusion, chromatography, absorption
chromatography ,ion exchange chromatography, hydrophobic interaction
chromatography and affinity chromatography. peptides are purified by
reversed-phase high-pressure chromatography. protein purity is assessed
by poly acryl amide gel electrophoresis .
Long amino acid polymers or polypeptides constitute the basic
structural unit of proteins, and the structure of a protein provides insight
into how it fulfills its functions. the edman reaction enabled amino acid
sequence analysis to be automated. mass spectrometry provides a
sensitive and versatile tool for determining primary structure and for the
identification of posttranslational modifications.DNA cloning and
molecular biology coupled with protein chemistry provide a hybrid
approach that greatly increases the speed and efficiency for determination
of primary structures of proteins. genomics—the analysis of the entire
oligonucleotide sequence of an organism’s complete genetic material has
provided
further
enhancements.
computer
algorithms
facilitate
identification of the open reading frames that encode a given protein by
using partial sequences and peptide mass profiling to search sequence
databases. scientists are now trying to determine the primary sequence
and functional role of every protein expressed in a living cell, known as
its proteome. a major goal is the identification of proteins whose
appearance or disappearance correlates with physiologic phenomena,
aging, or specific diseases.
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isoelectric focusing (ief):ionic buffers called ampholytes and an
applied electric field are used to generate a ph gradient within a
polyacrylamide matrix. applied proteins migrate until they reach the
region of the matrix where the ph matches their isoelectric point (pi), the
ph at which a peptide’s net charge is zero.
2. Proteins: Higher Orders of Structure
conformation versus configuration. proteins were initially classified by
their gross characteristics. the four orders of protein structure.
Peptide Bonds Restrict Possible, Secondary Conformations, types of
secondary structure.
the _ helix and the _ sheet.
the alpha helix
the beta sheet
loops & bends
helix-loop-helix motifs
tertiary & quaternary structure: the term “tertiary structure” refers to
the entire three dimensional conformation of a polypeptide.multiple
factors stabilize tertiary& quaternary structure three dimensional structure
is determined by x-ray crystallography or by nmr spectroscopy x-ray
crystallography
.the
native
conformation
thermodynamically favored.
protein folding
the native conformation of a protein
is thermodynamically favored
folding is modular
auxiliary proteins assist folding
chaperones
protein disulfide isomerase
proline-cis,trans-isomerase
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of
a
protein
is
several neurologic diseases.result from altered protein conformation.
Prions:The transmissible spongiform encephalopathies, or prion
diseases, are fatal neurodegenerative diseases characterized by
spongiform changes, astrocytic gliomas,and neuronal loss resulting from
the deposition of insoluble protein aggregates in neural cells. They
include Creutzfeldt-Jakob disease in humans, scrapie insheep, and bovine
spongiform encephalopathy (mad cow disease) in cattle. Prion diseases
may manifest themselves as infectious,Alzheimer’s Disease:Refolding or
misfolding of another protein endogenous to human brain tissue. collagen
illustrates the role
of, posttranslational processing in protein
maturation,protein maturation often involves making,& breaking covalent
bonds collagen is a fibrous protein Collagen is the most abundant of the
fibrous proteins that constitute more than 25% of the protein mass in
the human body.
Collagen Forms a Unique Triple Helix:
Collagen Is Synthesized as a
Larger Precursor
Nutritional & Genetic Disorders Can Impair.
Collagen Maturation.
Proteins may be classified on the basis of the solubility,shape, or function
or of the presence of a prosthetic group such as heme. Proteins perform
complex physical and catalytic functions by positioning specific
chemical groups in a precise three dimensional arrangement that is both
functionally efficient and physically strong.The gene-encoded primary
structure of a polypeptide is the sequence of its amino acids. Its
secondary structure results from folding of polypeptides into hydrogenbonded motifs such as the α helix, the β-pleated sheet, β bends, and loops.
Combinations of these motifs can form supersecondary motifs.Tertiary
structure concerns the relationships between secondary structural
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domains. Quaternary structure of proteins with two or more polypeptides
(oligomeric proteins) is a feature based on the spatial relationships
between various types of polypeptides. Primary structures are stabilized
by covalent peptide bonds. Higher orders of structure are stabilized by
weak forces multiple hydrogen bonds, salt (electrostatic) bonds, and
association of hydrophobic R groups.
Protein folding is a poorly understood process.Broadly speaking, short
segments of newly synthesized polypeptide fold into secondary structural
units. Forces that bury hydrophobic regions from solvent then drive the
partially folded polypeptide into a “molten globule” in which the modules
of secondary structure are rearranged to give the native conformation of
the protein. Proteins that assist folding include protein disulfide
isomerase,
proline-cis,trans,-isomerase,
and
the
chaperones
that
participate in the folding of over half of mammalian proteins. Chaperones
shield newly synthesized polypeptides from solvent and provide an
environment for elements of secondary structure to emerge and coalesce
into molten globules.
3.Proteins:Myoglobin & Hemoglobin
the heme proteins myoglobin and hemoglobin maintain a supply of
oxygen essential for oxidative metabolism.myoglobin, a monomeric
protein of red muscle,stores oxygen as a reserve against oxygen
deprivation.hemoglobin, a tetrameric protein of erythrocytes,transports
o2 to the tissues and returns co2 and protons to the lungs. heme & ferrous
iron confer the ability to store & to transport oxygen.myoglobin and
hemoglobin contain heme, a cyclic tetrapyrrole consisting of four
molecules of pyrrole linked by α-methylene bridges. Myoglobin Is Rich
in α Helix, Histidines F8 & E7 Perform Unique Roles in Oxygen
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Binding,The distal histidine,His E7, lies on the side of the heme ring
opposite to His F8.The Iron Moves Toward the Plane of the
Heme When Oxygen Is Bound.
Apomyoglobin Provides a Hindered
Environment for Heme Iron
the oxygen dissociation curves for myoglobin & hemoglobin suit their
physiologic roles.
the allosteric properties of hemoglobins result from their quaternary
structures,Hemoglobin Is Tetrameric:Hemoglobins are tetramers
comprised of pairs of two different polypeptide subunits. Greek letters are
used to designate each subunit type. The subunit composition of the
principal hemoglobins are α2β2 (HbA; normal adult hemoglobin), α2γ2
(HbF; fetal hemoglobin), α2S2 (HbS; sickle cell hemoglobin), and α2δ2
(HbA2; aminor adult hemoglobin). The primary structures of the β, γ, and
δ chains of human hemoglobin are highly conserved. Myoglobin & the _
Subunits of Hemoglobin Share Almost Identical Secondary and Tertiary
Structures.
Oxygenation
of
HemoglobinTriggers
Conformational
Changes
in the Apoprotein.
Termed cooperative binding,
P50 Expresses the Relative Affinities
of Different Hemoglobins for Oxygen
Oxygenation of Hemoglobin Is Accompanied by Large Conformational
Changes.
After Releasing O2 at the Tissues,Hemoglobin Transports CO2 &
Protons
to the Lungs.
The heme iron is ferric rather than ferrous. Methemoglobin thus can
neither bind nor transport O2. Normally, the enzyme methemoglobin
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reductase reduces the Fe3.+ of methemoglobin to Fe2,Methemoglobin
can arise by oxidation of Fe2+ to Fe3,
numerous mutant human
hemoglobins have been identified.methemoglobin & hemoglobin m,
hemoglobin s .biomedical implications myoglobinuria.
Anemias:Anemias, reductions in the number of red blood cells or of
hemoglobin in the blood, can reflect impaired synthesis of hemoglobin .
Thalassemias:The genetic defects known as thalassemias result from
the partial or total absence of one or more α or β chains of hemoglobin.
Over 750 different mutations have been identified, but only three are
common. Either the α chain (alpha thalassemias) or β chain (beta
thalassemias)can be affected.glycosylated hemoglobin (hba1c)
Myoglobin is monomeric; hemoglobin is a tetramer of two subunit
types (α2β2 in HbA). Despite having different primary structures,
myoglobin and the subunits of hemoglobin have nearly identical
secondary and tertiary structures. Heme, an essentially planar, slightly
puckered, cyclic tetrapyrrole, has a central Fe2 + linked to all four
nitrogen atoms of the heme, to histidine F8, and, in oxyMb and oxyHb,
also to O2. Hemoglobin also functions in CO2 and proton transport from
tissues to lungs. Release of O2 from oxyHb at the tissues is accompanied
by uptake of protons due to lowering of the pKa of histidine Residues.
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L3&4
1. Glycoproteins
glycoproteins
occur
widely&perform
numerous
functions
.oligosaccharide chains encode biologic information.techniques are
available for detection,purification,& structural analysis of glycoproteins.
eight sugars predominate in human glycoproteins , as sugar donors in
many biosynthetic reactions .exo- & endoglycosidases facilitate study of
asialoglycoprotein receptor is involved in clearance of glycoproteins.
certain glycoproteins from plasma by hepatocytes.lectins can be used to
purify glycoproteins & to probe their functions.there are three major
classes of glycoproteins.(1) those containing an O-glycosidic linkage
(ie,O-linked), involving the hydroxyl side chain of serine or threonine and
a sugar such as N-acetylgalactosamine (GalNAc-Ser[Thr]); (2) those
containing an N-glycosidic linkage (ie, N-linked), involving the amide
nitrogen ,of asparagine and N-acetylglucosamine (GlcNAc- Asn); and (3)
those linked to the carboxyl terminal amino acid of a protein via a
phosphoryl-ethanolamine moiety joined to an oligosaccharide (glycan).
glycoproteins contain several types of o-glycosidic linkages. Mucins
have a high content of o-linked oligosaccharides & exhibit repeating,
Amino Acid Sequences.The Biosynthesis of O-Linked Glycoproteins
uses nucleotide sugars.n-linked glycoproteins contain an asn-glcnac
linkage.complex, hybrid, & high-mannose are the three major classes of
n-linked oligosaccharides.the biosynthesis of n-linked glycoproteins
involves dolichol-p-p-oligosaccharide. assembly & transfer of dolichol-pp-oligosaccharide. processing of the oligosaccharide chain.
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The Endoplasmic Reticulum & Golgi Apparatus Are the Major Sites of
Glycosylation.Some
Glycan
Intermediates
Formed
During
N-
Glycosylation Have Specific Functions. Several Factors Regulate the
Glycosylation of Glycoproteins.Tunicamycin Inhibits N- but Not OGlycosylation.some proteins are anchored to the plasma membrane by
glycosylphosphatidylinositol structures. glycoproteins are involved in
many biologic processes & in many diseases.Glycoproteins Are
Important in Fertilization. Selectins Play Key Roles in Inflammation & in
Lymphocyte Homing.Abnormalities in the Synthesis of Glycoproteins
Underlie Certain Diseases.I-Cell Disease Results From Faulty Targeting
of Lysosomal Enzymes. Genetic Deficiencies of Glycoprotein Lysosomal
Hydrolases Cause Diseases Such as α-Mannosidosis.
Glycoproteins are widely distributed proteins—with diverse
functions—that contain one or more covalently linked carbohydrate
chains. The carbohydrate components of a glycoprotein range from 1% to
more than 85% of its weight and may be simple or very complex in
structure. At least certain of the oligosaccharide chains of glycoproteins
encode biologic information; they are also important to glycoproteins in
modulating their solubility and viscosity, in protecting them against
proteolysis, and in their biologic actions. The structures of many
oligosaccharide chains can be elucidated by gas-liquid chromatography,
mass spectrometry,and high-resolution NMR spectrometry. Glycosidases
hydrolyze specific linkages in oligosaccharides and are used to explore
both the structures and functions of glycoproteins.Lectins are
carbohydrate-binding proteins involved in cell adhesion and other
biologic processes. The major classes of glycoproteins are O-linked
(involving an OH of serine or threonine), N-linked (involving the N of
the amide group of asparagine), and lycosylphosphatidylinositol (GPI)linked.Mucins are a class of O-linked glycoproteins that are distributed
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on the surfaces of epithelial cells of the respiratory, gastrointestinal, and
reproductive tracts. The Golgi apparatus plays a major role in
glycosylation reactions involved in the biosynthesis of glycoproteins.The
oligosaccharide chains of O-linked glycoproteins are synthesized by the
stepwise addition of sugars donated by nucleotide sugars in reactions
catalyzed by individual specific glycoprotein glycosyltransferases.In
contrast, the biosynthesis of N-linked glycoproteins involves a specific
dolichol-P-P-oligosaccharide and various glycosidases. Depending on the
glycosidases and precursor proteins synthesized by a tissue,it can
synthesize complex, hybrid, or high-mannose types of N-linked
oligosaccharides. Glycoproteins are implicated in many biologic
processes. For instance, they have been found to play key roles in
fertilization and inflammation. A number of diseases involving
abnormalities in the synthesis and degradation of glycoproteins have been
recognized. Glycoproteins are also involved in many other diseases,
including influenza, AIDS, and rheumatoid arthritis. Developments in the
new field of glycomics are likely to provide much new information on the
roles of sugars in health and disease and also indicate targets for drug and
other types of therapies.
2. Plasma Proteins &Immunoglobulins
the blood has many functions: The functions of blood—except for
specific cellular ones such as oxygen transport and cell-mediated
immunologic defense—are carried out by plasma and its constituents
.Plasma consists of water, electrolytes, etabolites,nutrients, proteins, and
hormones. The water and electrolyte composition of plasma is practically
the same as that of all extracellular fluids. Laboratory determinations of
levels of Na+, K+, Ca2+, Cl−, HCO3 −, PaCO2,and of blood pH are
important in the management of many patients.
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plasma contains a complex mixture of proteins: The Concentration of
Protein in Plasma Is Important in Determining the Distribution of Fluid
Between Blood & Tissues.
Table. Major functions of blood.
(1) Respiration—transport of oxygen from the lungs to the tissues and of
CO2 from the tissues to the lungs
(2) Nutrition—transport of absorbed food materials
(3) Excretion—transport of metabolic waste to the kidneys,lungs, skin,
and intestines for removal
(4) Maintenance of the normal acid-base balance in the body
(5) Regulation of water balance through the effects of blood on the
exchange of water between the circulating fluid and the tissue fluid
(6) Regulation of body temperature by the distribution of body heat
(7) Defense against infection by the white blood cells and circulating
antibodies
(8) Transport of hormones and regulation of metabolism
(9) Transport of metabolites
(10) Coagulation
Plasma Proteins Have Been Studied Extensively
a. most plasma proteins are synthesized in the liver.
b. plasma proteins are generally synthesized onmembrane-bound
polyribosomes.
c. most plasma proteins are glycoproteins.
d. many plasma proteins exhibit polymorphism.
e. each plasma protein has a characteristic half-life in the circulation.
f. the levels of certain proteins in plasma increase during acute
inflammatory states or secondary to certain types of tissue damage.
Albumin Is the Major Protein in Human Plasma.Haptoglobin Binds
Extracorpuscular Hemoglobin, Preventing Free Hemoglobin From
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Entering the Kidney: Haptoglobin (Hp) is a plasma glycoprotein that
binds extracorpuscular hemoglobin (Hb) in a tight noncovalent complex
(Hb-Hp). The amount of haptoglobin in human plasma ranges from 40
mg to 180 mg .Absorption of Iron From the Small Intestine Is Tightly
Regulated:Transferrin (Tf) is a plasma protein that plays a central role in
transporting iron around the body to sites where it is needed.Iron
Deficiency Anemia Is Extremely Prevalent.Ferritin Stores Iron in Cells.
Hereditary Hemochromatosis Is Due to Mutations in the HFE Gene.
Ceruloplasmin Binds Copper, & Low Levels of This Plasma Protein Are
Associated With Wilson Disease.Copper Is a Cofactor for Certain
Enzymes. The Tissue Levels of Copper & of Certain Other Metals Are
Regulated in Part by Metallothioneins. Menkes Disease Is Due to
Mutations in the Gene Encoding a Copper- Binding P-Type
ATPase.Wilson Disease Is Also Due to Mutations in a Gene Encoding a
Copper-Binding P-Type ATPase.Deficiency of _1-Antiproteinase (_1Antitrypsin) Is Associated With Emphysema & One Type of Liver
Disease.α2-Macroglobulin Neutralizes Many Proteases & Targets Certain
Cytokines to Tissues Amyloidosis Occurs by the Deposition of
Fragments
of
Various
Plasma
Proteins
in
Tissues.plasma
immunoglobulins play a major role in the body’s defense mechanisms.All
Immunoglobulins Contain a Minimum of Two Light & Two Heavy
Chains All Light Chains Are Either Kappa or Lambda in Type The Five
Types of Heavy Chain Determine Immunoglobulin Class .Five classes of
H chain have been found in humans , distinguished by differences in their
CH regions. They are designated γ, α, μ δ, and ε. The μ and ε chains each
have four CH domains rather than the usual three. The type of H chain
determines the class of immunoglobulin and thus its effector
function.There are thus five immunoglobulin classes: IgG, IgA,IgM, IgD,
and IgE.No Two Variable Regions Are Identical.Property IgG IgA IgM
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IgD IgE The Constant Regions Determine Class-Specific Effector
Functions. Both Light & Heavy Chains Are Products of Multiple Genes
Antibody Diversity Depends on Gene Rearrangements Immunoglobulin
Major Functions.Class (Isotype) Switching Occurs During Immune
Responses. Both Over- & Underproduction of Immunoglobulins May
Result in Disease States.Hybridomas Provide Long-Term Sources of
Highly Useful Monoclonal Antibodies.The Complement
System
Comprises About 20 Plasma Proteins & Is Involved in Cell Lysis,
nflammation, & Other Processes.Plasma contains many proteins with a
variety of functions. Most are synthesized in the liver and are
glycosylated. Albumin, which is not glycosylated, is the major protein
and is the principal determinant of intravascular osmotic pressure; it also
binds many ligands, such as drugs and bilirubin. Haptoglobin binds
extracorpuscular hemoglobin,prevents its loss into the kidney and urine,
and hence preserves its iron for reutilization. Transferrin binds iron,
transporting it to sites where it is required. Ferritin provides an
intracellular store of iron. Ceruloplasmin contains substantial amounts of
copper,but albumin appears to be more important with regard to its
transport. Both Wilson disease and Menkes disease, which reflect
abnormalities of copper metabolism, have been found to be due to
mutations in genes encoding copper-binding P-type ATPases. α1Antitrypsin is the major serine protease inhibitor of plasma, in particular
inhibiting the elastase of neutrophils.Genetic deficiency of this protein is
a cause of emphysema and can also lead to liver disease.α2Macroglobulin is a major plasma protein that neutralizes many proteases
and targets certain cytokines to specific organs. Immunoglobulins play a
key role in the defense mechanisms of the body, as do proteins of the
complement system. Some of the principal features of these proteins are
described.
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