Factors that Affect Denaturation Proteins
Denaturing agents
1. physical factors
Temperature, pressure, mechanical shear force, ultrasonic vibration and ionizing radiation
causes the protein to lose its biological activity.
2. chemical factors
Acids and alkalis, organic solvents (actone, ethanol), detergents (cleaning agents), certain
amides urea, guandidine hydrochloride, alkaloids, and heavy metal salts (Hg, Cu, Ba, Zn,
Cd…) Cause the denaturation.
Properties of a Denatured Protein
A. an increase in number of reactive and functional group in the composition of the native
protein molecule ( side chain group of amino acids, COOH, NH2, SH, OH … etc)
B. Reduced solubility and pronounced propensity for precipitation
this occurs due to loss of the hydration shell and the unfolding of protein molecules with
concomitant exposure of hydrophobic radicals and neutralization of charged polar groups.
C. Configurational alteration of the protein molecule.
D. Loss of biological activity evoked by the disarrangement of the native structural molecular
organization.
E. Access of proteolytic enzymes in comparasion with the native protein
Clinical Application of Denaturation
The amounts of proteins found in the urine, serum, CSF are utilized to asses various
pathological conditions. The appearance of proteins like Albumin and Globulin in the urine can
be detected by precipitating them using ammonium sulphate. This could be used to asses the
degree of kidney impairment and glomerular permeability.
In some disease, abnormal proteins may be present in plasma and be filtered at the glomerule.
The most important member is Bence-jons’ protein which is most often associated with multiple
myeloma. So recognition of such protein in the urine may be useful in the diagnosis of the
disease.
This could be done by treating few ml of urine with few ml of hydrochloric acid giving a white
ring at the junction of the two fluids.
In the case of CSF protein estimation and analysis, a saturated phenol solution is used where 2
drops of CSF with 2ml of 10gm phenol dissolved in distilled water to check for turbidity. If the turbidity increases indicates an increase in Globulins. An increase in γ-globulins is observed in
case of multiple sclerosis and Neurosyphilis.
The normal level of Albumin in the CSF is 25-30mg%. When CSF treated with sulphosalisalic
acid with equal amounts and turbidity increases indicating an increase in the concentration of
Albumin in the CSF may be attributed to Acute meningities (usually about 35-40mg% when
measured spectrometrically).
Usually the level doesn’t exceed 1gm/L. only in the case of Multiple scleroma and spinal block
10gm/l might be observed.
Hemoglobin
Humans are aerobic organisms. Their lungs extract (O2) from inhaled gases.
The inspired (O2) leads to a more efficient utilization of fatty acids. Where as the expired CO2 is
a major product of cellular metabolism.
Living systems contain protein that interact with O2 and consequently increase its solubility in
H2O and sequester it for further reaction.
In mammals, Myoglobin (Mb) is found primarily in skeletal and striated muscle which mainly
serves as a store of O2 in the cytoplasm and deliver it on demand to the mitochondria.
Where as, Hemoglobin (Hb) is restricted to the Erythrocytes which is responsible for the
movement of O2 between lungs and other tissues .