Proteins Flashcards
How can we make more proteins than the 20000 genes which our human genome consists of ?
Because the genes can be spliced differently to produce different proteins
Also can chemically modify proteins at particular residues
How much of the dry weight of an organism is protein ?
50%
What residues in proteins can be phosphorylated ?
Ones which have a hydroxyl side chain
Eg serine-10, serine-16, tyrosine-6 and threonine-17
Aspartic acid and cysteine can also be phosphorylated
How would you determine which residue of phospholambam was phosphorylated in Vivo ?
Mouse provided with radioactive phosphate which combines with ATP
Mouse stimulated with beta agonist
Mouse is then killed at the appropriate time
Heart is removed immediately and frozen to preserve proteins
Proteins of the heart are dissolved by homogenisation in gentle non denaturants detergent
Solution also contain phopshatase inhibitors and magnesium chelates to prevent phosphorylation/dephosphorylation
Phospholambam can be separated using a specific antibody and then it can be sequenced and the sites of phosphorylation with fluoresce
Give examples when total serum protein concentration needs to be assessed ?
To look for nutritional abnormalities, kidney disease, liver failure
What is the normal levels of albumins and immunoglobulins in serum protein and what can a high or low serum protein concentration indicate ?
6-8.3g/dL or 600-830g/L
High- chronic inflammation of an infections(HIV, hepatitis c or b), multiple myeloma
Low- liver disease, malabsorption and agammaglobulinaemia
Why is total protein concentration of urine tested ?
Kidney diseSe, nephropathy in diabetes and several cardiovascular diseases
What is the Lowry assay ?
Technique for measuring total protein concentration by copper chelation by the protein
What is the Bradford assay ?
Technique for measuring total protein concentration by a dye binding assay
What amino acids absorb light in the wavelength of 280Nm and why ?
Tyrosine, tryptophan and phenylalanine
They have aromatic rings in their side chains which have delocalised electrons which can absorb the light
What is the molar extinction coefficient ?
It is a measure of how strongly a protein will absorb light at a particular wavelength
Can all proteins be detected equally with the A280 technique ?
No
Because it is determined by the frequency of tyrosine, tryptophan and phenylalanine residues in the protein
Can all proteins be detected equally using the Bradford assay ?
No
Because the dye used in this assay which is coomassie blue only binds to positively charged residues in an acidic environment
Mainly detects arginine, histidine and lysine
Can all proteins be detected equally in the Lowry assay ?
Yes
Because it is based on copper chelation of the backbone so it doesn’t rely on the different residues in a protein
What features are unique to particular proteins and can be exploited for extraction ?
Primary sequence
Ligand binding sites
Antibody binding sites
Unique spectral feature