Module 6: Amino Acids and Proteins Obj. 6 Flashcards
reaction of the Biuret method for total protein
peptide bonds in protein + cupric ions at an alkaline pH = violet color complex
Purpose of potassium iodide in Biuret method
prevents auto-reduction of Cu++ to Cu+
Purpose of sodium-potassium-tartrate in Biuret method
maintain Cu++ in solution at alkaline pH
Preferred type of sample for Biuret method
serum
Which samples are not suitable for Biuret method? Why?
urine - ammonia present interferes with test
CSF - method is not sensitive enough
uses for dye-binding methods
albumin, urine total protein and electrophoretic quantitation
requirements of dye-binding methods
two separate wavelengths for measuring absorbance of the free dye and the dye-protein complex
Minimal interfering substances (ex. Hb, bili, lipemia)
Constant temp
Principle of protein electrophoresis
separation of charged molecules on support medium in an electrical field
Proteins in alkaline solution have net neg. charge
what does pH determine in EP
the state of ionization of the charged groups of the constituent amino acids of proteins and therefore their electrical charges
what does ionic strength determine in EP
resolution and mobility
buffer typically used in serum EP? What is its pH?
Barbital based buffer
pH 8.6
anions, cations, anode, cathode definition
anions are negatively charged ions that move towards the anode (pos pole)
cations are positively charged ions that move towards the cathode (neg pole)
which form are proteins in and which pole do they migrate towards in typical protein EP
proteins will have negative charge (anions) and will move towards anode (pos pole)
Effect on mobility and resolution of low ionic strength buffers
faster migration
low resolution
Effect on mobility and resolution of high ionic strength buffers
slow migration
high resolution
what is electroendosmosis and how does it occur
movement of buffer fluid itself under the influence of applied voltage in the electric field
Water molecules in close association with medium become pos charged and are attracted towards the cathode
Movement of water carries some proteins backwards
Gamma region
ohms law
voltage (V) = Current (I) x Resistance (R)
effect on migration from constant current
Constant current: stabilizes migration rate
5 bands produced in serum EP
Albumin Alpha 1 alpha 2 beta gamma
⍺ 1 band contains
⍺1-fetoprotein
⍺1-antitrypsin
⍺1-lipoproteins
Thyroid binding globulin
⍺2 band contains
Haptoglobin
Ceruloplasmin
⍺2-macroglobulin
Pre-Beta-lipoprotein
Beta-globulin contains
Transferrin Hemopexin B-lipoprotein B2-microglobulin C3 and C4 CRP Free Hb
gamma-globulin band contains
immunoglobulins IgG, IgM, IgD, IgE
2 primary functions of albumin in the body
transport of substances in plasma
Regulating osmotic pressure and fluid balance in the extracellular compartment
