LAB - Kinetic Spec & Protein Electrophoresis Flashcards
higher level functioning enzyme results in…
higher rxn raes
y-Glutamyltransferase (y-GT)
- catalyzes transfer of glutamyl group from a glutamyl peptide to an AA of another peptide
- present in several organ systems
- increase in serum = from hepatobiliary system
- used in diagnosis of liver diseases such as alcoholic cirrhosis and primary + secondary liver tumors
measure of this enzyme may also be used to confirm if an increase in ALP originates in the liver
y-GT
formation of this product is proportional to y-Gt activity and is measured kinetically at this wavelength
- 5-amino-2-nitrobenzoate (end product)
- 405 nm
Kinetic Assays (2)
- enzyme incubated with substrate and substrate product
- co-factor measured continuously or at several points as a function of time
this assay allows for the demonstration of the linearity of a rxn
Kinetic Assay
- usually measured over a very short period of time = little danger of enzyme inactivation
lag phase in SOP for kinetic enzyme assay
incubation of serum with reagent (4 mins)
linear phase
- where constant amount of product is generated per unit time
- enzyme activity measured in this phase
- corresponds to zero order kinetics
substrate depletion phase
- plateau
- change in absorbance wouldn’t change as much as [products] would stay stagnant
why are we not using standards for the kinetic enzyme assay?
we are measuring enzyme activity! unknown for each patient so a standard would not really tell us what to “aim” for
- standards are essentially used to determine concentrations of unknowns
the concentration or activity of ions in a solution or buffer
ionic strength
- sum of the concentrations of all ions in a solution, weighted by the squares of their charges
what does ionic strength depend on?
both the concentration of each ion and the square of its charge
NOTE: in mixtures, the degree of dissociation must be considered to determine the actual concentration of ions
these dissociate completely
salts, strong acids, and bases
for univalent electrolyte solutions, such as NaCl, these are the same
the ionic strength and concentration (in mol/L)
ionic strength takes into account the number of charges
- complete ionization of multiple charge ions = ionic strength greater than the concentration
- ionic strength is 3 times the concentration with divalent ions; 6 times with trivalent
ionic strength of sucrose
0 because does not have any ions (non-ionizable)
these determine what is ionizable
buffers
- salts will completely ionize; only free ions contribute to ionic strength
- acids will ionize according to their dissociation constant but will contribute much less than salts
rate of migration in electrophoresis is dependent on
- net charge of molecule
- size and shape of molecule
^ MAIN - type + ionic strength of buffer
- buffer pH
- strength of current
- migration distance is increased w time (longer time also increases diffusion)
- temp and evaporation
- electroendosmosis
when pH of the buffer is greater than pI of proteins =
net charge is negative
- when pH is less than pI = pos
T or F. the greater the charge on the protein, the greater than attraction to the electrode
T
T or F. as ionic strrength increases, mobility increases as well
F! mobility decrease
- buffer ions surround the charged molecules and hinder their movement = giving sharper bands/better resolution (increased heat from increased current is undesirable)
- lower ionic strength = greater migration but with some loss of resolution
what happens to ionic strength if buffer has been used too often?
ionic strength increases
buffer pH
- affects net charge of protein
- the greater the difference between the pI of the protein and the pH, the greater the magnitude of the charge
- pH must be such that it is possible to separate the proteins in a mixture, but not denature them
strength of current in electrophoresis
movement is proportional to field strength (i.e. increased current causes faster migration)
temperature and evaporation in electrophoresis
- as temp increases = evaporation increases
= drying of the strips - drying causes the buffer to rise into the strip from buffer chambers via the wick (wick flow) and will influence protein mobility
what is electroendosmosis?
- electrophoretic support medium in contact with water takes on a negative charge bc of the adsorption of OH- ions; immobile
- positive ions (H3O+) cluster about the fixed neg charge sites forming an ionic cloud of mostly positive ions
- current applied = movement of ionic cloud results in movement of buffer as well and this movement of solvent + solutes = electroendosmosis
- if molecules have a pI near the pH of buffer = immobile or swept back toward cathode
- strong electroendosmosis = paper, agar gel = y-globulins are swept behind the line of application
when is the electroendosmosis effect greater?
when ionic strength of the buffer is higher (more ions)
