Unit 3 - Protein Flashcards
Primary Structure
Amino acid sequence determined by DNA
Secondary structure
more complex a.a. chain
Tertiary structure
Complex folding, 3D structure
Quaternary Structure
More than on protein chain
When is biological activity lost in a protein?
When secondary/tertiary/quaternary structure denatured
Simple protein
No other major chemical class associated with them
Conjugated proteins
Nonamino acid chemical associated with protein
(prosthetic group)
Lipoprotein
proteins with fat
(cholesterol, triglyceride, phospholipids)
Metalloproteins
Contain a metal
(hemoglobin)
Glycosylated proteins
Contain carbohydrate
(HgbA1c)
Types of measurement methods
Light absorption
Light refraction
Dye bonding/Colorimetric
Electrophoresis
215nm
Peptide bond
280 nm
Aromatic rings present in a.a.
What proteins are detected at 280 nm?
Tyrosine
Tryptophan
Phenylalanine
What measurement method is best for a known or pure protein?
Light absorption
What measurement method is nondestructive for proteins?
Light absoprotion
What is a total protein measurement that is rapid?
Light refraction
What specimen is worst for light refraction?
CSF
Low protein specimen
What are the physical properties used to detect and measure proteins
Molecular size
Electrical charge
Solubility
What affects electrical charge?
pH
How does solubility measure proteins
A protein is dissolved or precipitated out of a mixture
What is used to manipulate solubility
SUlfosalicylic acid
What does increased turbidity mean for a solubility sample?
More protein was precipitated out
What are the major components of biuret?
Sodium Hydroxide + Copper sulfate
What dye bonding test is used to measure total protein
Biuret
What dye bonding test is used to measure albumin?
BCG
BCP
How does biuret work to bond to the protein?
Copper ions react with carbonyl oxygen and amide nitrogen in peptide bond
How many peptide bonds are required for biuret analysis?
At least two
Are dipeptides measured by biuret ?
No, two amino acids with one peptide bond
What is a positive color change for biuret?
Blue biuret makes a violet color with protein
Color intensity increases as ___ increases
Peptide bonds amount
Total protein reference range in serum
6-8 g/dL
What’s the difference in tp concentration from serum and other fludis
Serum has highest concentration, others are measured in mg or ng quantities
When would a pink color be produced in biuret method
In presence of small peptides
How is biuret calibrated?
With albumin alone because other protein concentrations are too diverse and unpredictable
What samples are not appropriate for biuret
CSF
Urine
What dye is used to measure lower protein levels like in CSF or Urine
Coomasie Blue
Ponceau S
Amido black
Pyrogallol red
When is insensitivity in dye bonding for globulins NOT a problem?
A leaky glomerulus in the kidney because albumin spills into urine
When is insensitivity in dye ending for globulins a problem?
Multiple myeloma because free light chains spill into urine
Which test would you choose for clinical decisions based on serum albumin? BCG or SPE?
BCG/BCP
Total Protein formula
Alb + Glob
Globulin formula
TP - Albumin
A/G ratio
Albumin divided by globulin
Albumin reference range in serum
3.5-5 g/dL
Globulins reference range in serum
2.5-3 g/dL
Protein reference range in urine
1-14 mg/dL
<100 mg/24 hours
Protein reference range in CSF
15-45 mg/dL
Note small window
Calculation of protein in urine using 24 hours specimen
(given total protein mg/dL divided by 100mL) = (xmg/given total mL)
What urine specimen eliminated biological variation and dilution effects?
24 hour urine
Why would 24 hour urine be required?
Evaluate urine and eliminate biological variation and dilution effects
What is the point where the number of positive charges equal the number of negative charges?
Isoelectric point
If the isometric point of albumin is 4.6, what is its charge at 4.6?
No net charge
Neutral
Albumin Iso point is 4.6, what is its charge at 5.1
Positive charge
What is the isoelectric point of globulins
5.1-6.2
Which protein has the highest net negative charge at a buffer pH of 8.6
Albumin
What is the usual pH of the buffer for SPE
8.6
Anode
Positive pole
Cathode
Negative pole
Where do anions move?
To the anode
Where do cations move?
To the cathode
The more the negative charge, the __ the movement?
faster
What causes the visible application point on SPE?
Precipitated protein from a thawed specimen or debris
A molecule has no charge, where will it move in an electrical field?
Nowhere
What does the buffer make the molecules do?
Take on a negative charge to move towards the anode
Where is the origin of the application point?
Cathode, negative pole
Fast equivalent to
Positive end
Slow equivalent to
Negative end
Frictional coefficient
Medium resists particle movement
how do larger, more complex molecules move if the pores in the medium are small?
Slowly
If the support medium has small pores, what is separation based on?
Size and charge
If the support medium has large pores, what Is separation based on?
Charge only
High voltage can cause
High temps that denature proteins
Usually, the slab or room should be…
Cold
What is the standard buffer for SPE?
Sodium barbital required for electrons to flow
What kind of ionic strength do you want your buffer to have?
Low, the extra ions will impede migration by adding charge
What is wick flow caused by
Evaporation of the buffer
What does wick flow look like
Proteins make a trail
How do you fix wick flow
Cool the system or buffer
Keep lid on electrophoresis chamber
Change buffer/use new buffer every time
What is electroendosmosis
Weak anions bump into strong cations and move behind the application point
Tracer dyes
Stick to albumin and make the band look fat
Two albumin bands can be caused by
Genetic bisalbuminemia
or
Drugs
Why would drugs cause two albumin bands
They can bind to the albumin
Split beta bands have
Transferrin and C3
If a split beta gel wasn’t being used, what would the C3 band make you think?
If the peak is a monoclonal spike
or
If specimen is fresh
What does fresh specimen look like eon SPE?
Monoclonal spike in beta band, split beta band
What does fibrinogen do to SPE
Looks like a spike if plasma used
Where does fibrinogen migrate to on SPE
Between beta and gamma
How to solve the spike caused by fibrinogen?
Allow serum to fully clot
Add thrombin so blood will clot (if can’t be recollected)
Where does hemolysis appear on SPE?
Between a2 and B
What Ig are most commonly seen on SPE?
G, A, M
Why aren’t IgD or IgE seen on SPE?
Too low concentration, unless monoclonal
Where does CRP appear on SPE
Gamma region toward beta side
When would CRP be elevated on SPE?
Acute phase reactant
May mimic monoclonal spike
Isoelectric focusing
Electrophoresis done in pH gradient
How does isoelectric focusing work
Proteins stop moving when they reach the pH of their isoelectric point
