lab 4: lysozyme isolation & purification

what are the three objectives of the lab?

1. preparation of buffered egg white (BEW)

2. lysozyme assay with BEW

3. purify a protein using ion exchange chromatography

what is the function of a lysozyme?

damages bacterial cell walls by cleaving the mucopolysaccharide via hydrolysis, protect cells from bacteria infection

what is the most concentrated source of lysozymes?

egg white of chicken egg

what are the two applications of protein purification?

1. pharmaceuticals

2. nutrition

what is an example of pharmaceutical application of protein purification?

antibodies & vaccines

what is an example of nutrition application of protein purification?

digestive enzymes

what is ion exchange?

reversible electrostatic binding of ions to an insoluble, chemically inert matrix (resin)

what is anion exchange?

positively charged resin binding to negatively charged ions (anions)

what is cation exchange?

negatively charged resin binding to positively charged ions (cations)

what is pI?

the pH at which a particular protein has a net charge of zero

what does it mean when the buffer pH is greater than pI?

protein has net negative charges (right of the titration curve)

what does it mean when the buffer pH is less than pI?

protein has net positive charges (left of the titration curve)

what is the main egg white protein?

ovalbumin at 54%

should you allow the column to dry out?

NO

what charge is one the carboxylmethyl-sephadex beads?

negative

what is the pH for the Tris buffer?

8.2

what charge is one most of the proteins in BEW?

negative

will the BEW bind to the column? why?

no because the lysozyme is positively charged

what is the pH of the CAPS buffer?

10.5

what is the pI and pH for lysozyme with the CAPS buffer?

both 10.5, no charge

what is the filtrate in this experiment?

BEW

dilution factor definition

final total volume/original sample volume

what absorbance will we measure at?

280 nm

what is the goal of enzyme purification?

to separate the desired enzyme from a crude mixture of cellular components

what are the four consideration prior to enzyme purification?

1. the source

2. purification methodology

3. an enzyme assay

4. a method to measure protein present

what is meant by source availability?

easier to obtain larger quantities of the enzyme to isolate the enzyme

at what point does the lysozyme cleave the bacterium Micrococcus lysodeikticus?

1,4 linkage between N-acetylmuramic and N-acetylglucosamine

how is lysozyme activity measured?

spectrophotometrically by changing in light scattering of the bacterial suspension at 600 nm. intact cells scatter light but less light when they are broken

how is the magnitude of decreasing in light scattering used?

to measure the enzyme activity present in a given enzyme preparation

optic density (OD) definition

light scattering measured by a spectrophotometer

how many amino acids are in lysozymes?

129

why is carboxylmethyl-sephadex a cation exchanger?

because the carboxyl attached to the cellulose beads has a negative charge and binds cations

how is the negative charge of the carboxyl neutralized in the CM-sephadex beads?

by being in sodium form

what must the pH of the initial buffer system be and why?

must be above 6 for the carboxyl to be substantially protonated and negatively charged since the pKa of carboxyl is 4.2

what isoelectric points are most proteins at?

less than 7

what will be determined by the end of the experiment?

• protein concentration

• total protein

• enzyme activity

• specific activity

• total activity

• purification factor and yield

why will lysozymes be eluted from the column when an elution buffer with a pH of 10.5?

because the pH of the elution buffer is the same as the lysozyme isoelectric point of 10.5

at what absorbance will the fraction be measured to monitor elusion of the non-binding proteins?

280 nm

why will we use Tris Buffer to wash the column?

because the egg whites proteins do not carry a positive charge at a pH of 8.2