LAB - Kinetic Spec & Protein Electrophoresis

Question 1

higher level functioning enzyme results in…

Answer 1

higher rxn raes

Question 2

y-Glutamyltransferase (y-GT)

Answer 2

• 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

Question 3

measure of this enzyme may also be used to confirm if an increase in ALP originates in the liver

Answer 3

y-GT

Question 4

formation of this product is proportional to y-Gt activity and is measured kinetically at this wavelength

Answer 4

• 5-amino-2-nitrobenzoate (end product)

- 405 nm

Question 5

Kinetic Assays (2)

Answer 5

• enzyme incubated with substrate and substrate product

- co-factor measured continuously or at several points as a function of time

Question 6

this assay allows for the demonstration of the linearity of a rxn

Answer 6

Kinetic Assay

- usually measured over a very short period of time = little danger of enzyme inactivation

Question 7

lag phase in SOP for kinetic enzyme assay

Answer 7

incubation of serum with reagent (4 mins)

Question 8

linear phase

Answer 8

• where constant amount of product is generated per unit time
• enzyme activity measured in this phase
• corresponds to zero order kinetics

Question 9

substrate depletion phase

Answer 9

• plateau

- change in absorbance wouldn’t change as much as [products] would stay stagnant

Question 10

why are we not using standards for the kinetic enzyme assay?

Answer 10

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

Question 11

the concentration or activity of ions in a solution or buffer

Answer 11

ionic strength

- sum of the concentrations of all ions in a solution, weighted by the squares of their charges

Question 12

what does ionic strength depend on?

Answer 12

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

Question 13

these dissociate completely

Answer 13

salts, strong acids, and bases

Question 14

for univalent electrolyte solutions, such as NaCl, these are the same

Answer 14

the ionic strength and concentration (in mol/L)

Question 15

ionic strength takes into account the number of charges

Answer 15

• 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

Question 16

ionic strength of sucrose

Answer 16

0 because does not have any ions (non-ionizable)

Question 17

these determine what is ionizable

Answer 17

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

Question 18

rate of migration in electrophoresis is dependent on

Answer 18

• 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

Question 19

when pH of the buffer is greater than pI of proteins =

Answer 19

net charge is negative

• when pH is less than pI = pos

Question 20

T or F. the greater the charge on the protein, the greater than attraction to the electrode

Answer 20

T

Question 21

T or F. as ionic strrength increases, mobility increases as well

Answer 21

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

Question 22

what happens to ionic strength if buffer has been used too often?

Answer 22

ionic strength increases

Question 23

buffer pH

Answer 23

• 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

Question 24

strength of current in electrophoresis

Answer 24

movement is proportional to field strength (i.e. increased current causes faster migration)

Question 25

temperature and evaporation in electrophoresis

Answer 25

• 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

Question 26

what is electroendosmosis?

Answer 26

• 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

Question 27

when is the electroendosmosis effect greater?

Answer 27

when ionic strength of the buffer is higher (more ions)