Protein Biochemistry and Hemoglobin Ebook Flashcards
A very effective way to determine the molecular weight of a protein, the number of subunits in a purified protein, and to visualize many proteins simultaneously
SDS-PAGE
One problem with antibody affinity chromatography is that sometimes the only way to elute the proteinmay be under harch conditions, resulting in a
Denatured, inactive protein
A recombinant fusion protein is generated, in which an epitope is appended at either the N- or C-terminus of the coding region of the protein of interest. The proteins expressed can be easily identified in whole cell extracts and can be purified by immunoprecipitation using antibodies against the epitope
Epitope tagging
Eliminates the need for conventional methods of protein purification and animal immunization to generate antibodies, which are often more tedious and time-consuming
Epitope tagging
A plate-based assay designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones.
Enzyme-Linked Immunosorbent Assay (ELISA)
Makes it possible to visualize the distribution and localization of specific cellular components within a cell or tissue.
Immunohistochemistry (IHC)
Contain small amounts of purified proteins, which allow simultaneous determination of a great variety of analytes from small amounts of samples within a single experiment
Protein Microarrays (protein chips)
Heme is considered to be a non protein molecule that is required for biological activity. In other words, Heme is a
Cofactor
A monomeric protein (single polypeptide chain) with one heme group. It is found in muscle where it binds oxygen tightly until an oxygen-depleted state induces its release for metabolic oxidation.
Myoglobin
A tetrameric protein with four polypeptide chains (two α-globins and two β-globins). It is found in high concentration in red blood cells and servers to
transport oxygen from the lungs throughout the body.
Hemoglobin
The higher P50, the
Lower the affinity for oxygen
Binding at one site affects the activity of an enzyme (or a transport protein) at another site.
Allostery
Oxyhemoglobin does not bind
2,3-BPG
Picked up along with oxygen in the lungs and delivered to tissues
Nitric Oxide
Can be caused by exposure to oxidizing agents or by mutations in the hydrophobic pocket of Hb
Methemoglobinemia
Two proteins are involved in keeping levels of methemoglobin in our blood down: cytb5 reductase reduces cytb5 which in turn reduces
Methemoglobin
One of the major roles of fetal hemoglobins is to extract oxygen from the
-the reason it has ahigher affinity for oxygen than HbA
Maternal circulatory system in the placenta
This change in affinity of HbF relative to HbA is due to a histidine to serine mutation in the
BPG binding site
Glycation of the amino-terminus will remove a positive charge which will reduce affinity for
BPG
Absence or decreased synthesis of functional α-globin or β-globin chains. These can be caused by splicing defects, deletions of genes, or altered regulatory elements.
-Loss of function mutations
Thalassemias
Homozygous state with 2 defective genes
Thalassemia major
Heterozygous state with 1 normal and 1 defective gene
Thalassemia minor
In cases where α-globin gene expression is below 50% of normal, the β-globin forms β4 (HbH) tetramers which precipitate in red blood cells resulting in
Anemia
Mutations have been found to cause disease through one of what four different effects on protein function?
- ) Loss of function
- ) Gain of function
- ) Heterochronic expression
- ) Ectopic expression
The expression of a gene at the wrong time
Heterochronic expression
The expression of a gene in the wrong place
Ectopic expression
A loss of function due to deletion, leading to a reduction in gene dosage, is exemplified by the
α-thalassemias
Many other types of mutations such as a premature stop codon or of a missense or other mutation can also lead to a complete loss of function. All of these classes of mutation, and others, are illustrated by the
β-thalassemias
A group of hemoglobinopathies that result from a reduction in the abundance of β-globin, one of the major adult hemoglobin proteins in red blood cells
β-thalassemias
Missense mutation that locks hemoglobin in its high oxygen affinity state, thereby reducing oxygen delivery to tissues.
-Example of a gain of function mutation
Hemoglobin Kempsey
Some mutations in hemoglobin regulatory elements lead to the continued expression in the adult of the γ-globin gene, which is normally expressed at high levels only in fetal life. Such γ-globin gene mutations lead to a
phenotype called the
Hereditary persistence of fetal hemoglobin (HPFH)
Thalassemias due to reduced or absent production of a globin mRNA because of deletions or mutations in regulatory or splice sites of a globin gene reduce
Transcription
Thalassemias due to nonfunctional or rapidly degraded mRNAs with nonsense or frameshift mutations reduce
Translation
The most common single-gene diseases in humans, and they cause substantial morbidity
Hemoglobinopathies
Required for the gene expression of all genes in the β-globin cluster on chromosome 11
The locus control region (LCR)
What is more likely to cause a disease, mutation of a β-globin gene, or mutation of an α-globin gene
β-globin gene mutation
Why is a beta globin gene mutation more likely to result in a mutation?
A single β-globin gene mutation affects 50% of the β chains, whereas a single α-chain mutation affects only 25% of the α chains
Because α chains are the only α-like components of all
hemoglobins 6 weeks after conception, α-globin mutations cause severe disease in
both
Fetal and postnatal life
The hereditary disorders of hemoglobin can be divided into which three broad groups?
- ) Structural variants
- ) Thalassemias
- ) Hereditary persistence of fetalhemoglobin
Alter the globin polypeptide without affecting its rate of synthesis
Structural Variants
Decreased synthesis (or, rarely, extreme instability) of one or more of the globin chains, resulting in an imbalance in the relative amounts of the α and β chains
Thalassemias
Most variant hemoglobins result from
Point mutations
The hemoglobin structural variants can be separated into which three classes?
- ) hemolytic anemia
- ) Altered oxygen transport
- ) Varients that cause Thalassemia
Due to a beta chain substitution of Glutamate 6 for lysine
Hemoglobin C
Less soluble than HbA and tends to crystallize in red blood cells
HbC
α-Thalassemias involve mutations that prevent the alpha subunits from being synthesized. as a result, be get tetrameric beta subunits such as
- ) Fetal γ4 (Hb Bart’s)
2. ) β4 (HbH)
Fetal γ4 (Hb Bart’s) and β4 (HbH) are not capable of
Veleasing oxygen
Infants with severe α-thalassemia and high levels of Hb Bart’s suffer severe intrauterine hypoxia and are born with massive generalized fluid accumulation, a condition called
Hydrops fetalis
The onset of β-thalassemia is not apparent until a few months after
Birth
β-thalassemia results in hemoglobin being made up of four
Alpha subunits (leads to RBC destruction)
In contrast to α-thalassemia, the β-thalassemias are usually due to single-base pair substitutions rather
than to
Deletions
When the β-thalassemia alleles allow so little production of β-globin that no Hb A is present, the condition is designated
β0 thalassemia
What are some effective methods for treating βthalassemias?
Blood transfusion, Iron chelators, Bone marrow transplant
Carriers of one β-thalassemia allele are clinically well and are said to have
Thalassemia minor
Almost every type of mutation known to reduce the synthesis of an mRNA or protein has been identified
as a cause of
β-thalassemia
The majority of β-thalassemia patients with a decreased abundance of β-globin mRNA have abnormalities in
RNA splicing
The only disease-modifying therapy aproved for sickle cell anemia
Hydroxyurea
Increases total and fetal hemoglobin in children with sickle cell
Hydroxyurea
The change in expression during development of the various globin genes
Globin switching
Embryonic globin synthesis occurs in the
Yolk sac from 3rd to 8th week
At about the 5th week, the major site of hematopoiesis begins to move from the yolk sac to the
Fetal liver
-Resulting in HbF
What is the make-up of HbA2?
α2δ2
The expression of the β-globin gene has been found to be only partly controlled by the promoter and two enhancers in the immediate flanking DNA. Another critical element has been identified as the
Locus Control Region
What are two hemoglobinopathies that fall under the structural variant realm of hemolytic anemia?
Sickle Cell Anemia and HbC
Oxygenated Hemoglobin C tends to
Crystallize
Under deoxygenated conditions, sickle hemoglobin, HbS is only
1/5 as soluble as normal Hb
Infants with severe α-thalassemia and high levels of Hb Bart’s suffer severe intrauterine hypoxia and are born with massive generalized fluid accumulation, a condition called
Hydrops Fetalis
Often result in hypochromic, microcytic red blood cells
Thalassemias
The diagnosis of thalassemia minor can be supported by hemoglobin electrophoresis, which generally reveals an increase in the level of
HbA2
Characterized as β-thalassemia where β alleles allow so little production of β chains that there is no functional HbA
β0-thalassemia (a thalassemia major)
How many functional α chains are there in Hb Bart’s?
0
How many functional α chains are there in HbH (moderately severe hemolytic anemia)?
One
The diagnosis of thalassemia minor (heterozygous) can be supported by hemoglobin electrophoresis, which generally reveals an increase in the level of
Hb A2 (α2δ2)
Some deletions within the β-globin cluster do not cause thalassemia but rather a fascinating phenotype termed the
Hereditary persistence of fetal hemoglobin
One group of defects, which accounts for the great majority of patients, impairs the production of β-globin alone and causes
Simple β-thalassemia
What causes the complex β-thalassemias?
The β-glob in gene, as well as one or more other genes, or the LCR in the β-glob in cluster is removed
Lead to a decrease in the abundance of the β-globin mRNA and include promoter mutants, RNA splicing mutants (the most common), mRNA capping or tailing mutants, and frameshift or nonsense mutations that introduce premature termination codons within the coding region of the gene.
Mutations causing simple β-thalassemia so
Mutations in the β-globin gene that cause premature stop codons cause
β0 thalassemia
Mutations resulting in defects to the 5’ cap or 3’ tail of β-globin mRNA cause
β+-thalassemia
Start approximately 50 to 100 kb upstream of the β-globin gene cluster and extend 3’ to varying degrees
-Cause εγδβ- thalassemia
Deletions that remove the β-globin LCR
Given to children with sickle cell to prevent pneumococcal disease
Penicillin Prophylaxis
For HbSS (sickle cell), what affect do the following have on sickle cell aggregation?
- ) Increased BPG
- ) Low pH
- ) Glycosylation
- ) Increased sickling
- ) Increased sickling
- ) Decreased sickling
In HbSS, anything that stabilizes the T-state (decreases affinity for oxygen) will
Increase Sickling
If we have an individual that is heterozygous for beta thalassemia and heterozygous for sickle cell
Sickle cell band will be thicker than HbA band
If we have an individual that is heterozygous for alpha thalassemia and heterozygous for sickle cell
Sickle cell band will be thinner than HbA
