>50 AA (connected by peptide bonds) to be considered a protein,
can hydrolyze proteins. eg add 6 M HCl, reflux. heat solution to accelerate reaction, condenser to prevent loss of product by evaporation. hydrolyze to respective monomeric units (aa’s)
proteins: can be one polypeptide chain, or 2 or more 1’ aa sequence
2’ alpha-helix, beta-sheets
3’ defined function; tetramer, etc. every monomer is a single polypeptide proteins are amphoteric: can act as acid/base (electron acceptor/donor) zwitterion: #positively ch’d grp = # negatively ch’d group. 0 net charge. at different conditions, you can achieve zwitterionic form isoelectric pH consider all ionizable sidechains when determining pI.
R groups det/dictates protein property. eg hydrophobic vs hydrophilic R groups.
EXPT GOAL: isolate proteins, albumin (egg white) and casein (milk) general steps: extraction, isolation, purification
diffn8:
extraction goal is to destroy cellular components / take it from intracellular components.
disrupt cell membrane. cell membrane of plants, animals, bacteria….
if from animals: expect CM to be soft. possible methods: freezing and thawing, mechanical method (stirring, using mechanical beater(?), s_?)
if from plants: expect rigid (w/ CW)
may use mortar and pestle, add sand and grind; liquid N, freeze and pound
burst cell, release protein in extraction.
isolation: remove other cellular components.
2 methods: centrifugation, fractional precipitation (in expt).
centrifugation : basis of varying density. gravity has something to do w density (lightwt sa taas supposedly). kinds of centrifuge. larger radius, larger centrifugal force experienced
maximum speed, 10 mnutes etc. max out lang.
after centrifugation: fractional pption. 4 methods to form ppt. 1. isoelectric pption
a. control pH of system. @pI, 0 net charge, and thus insoluble in water. react w/ one another; aggregate/ppt out. (solubility: same IMFA to be soluble)
2. thru temperature change
a. proteins clump together at colder temperatures. deactivate protease (w/c cleave, denature proteins). 3. lowering of dielectric constant
a. add organic solvents (eg ethanol) to solution. polarity of solution decreases. proteins are CHARGED
biomolecules; decrease polarity, decreased solubility. EtOH is less polar than water. if u add more EtOH, bababa polarity ng buong solution (reduce prot-water intxn)
4. addition of salt
a. salt is ionic: instead of protein-water intxn, inaagawan ng salt. salt-water intxn prevalent.
purification
traces of other components still present.
exptal: chromatographic techniques (separation techniques); SDS-PAGE (can cut bands, melt gel);
SDS-PAGE
earlier: washed w destaining soln until clear, viewed in lightbox. / / / / // / / / / / / / / / / / / / /
protein of interest: albumin. produced in liver. when low body level of
albumin, indicates you have liver/kidney disease. liver disease: liver canno;;; kidney: excrete albumin
albumin can be found in serum. serum is matrix of blood. albumin uses: mainly for transpo of fats
add albumin to solution to stabilize enzyme? albumin interacts with antibodies, so that the enzymes don’t. “first line of defense”
pI of BSalbumin ~4.6, MW (BSA) 66.4 kDa
in expt: want egg white only. bc egg yolk is fatty. hassle iremove yolk. albumin content is also higher in egg white anyway. then, added HOAc to (disrupt cell mem), but mainly added bc we want to ppt out OTHER cellular components with low pI. (lowers pH of solution).
next step: filtration thru cheesecloth. remove unwanted proteins. only
albumin in filtrate hopefully. add ammonium sulfate: salting out; trying to ppt proteins thru salting out effect. add equiv vol of soln. ammonium sulfate, why? cheap, high ionic character, effective disruptor of protein-water intxns. discard ppt. then add ammonium sulfate again, centrifuge, then collect ppt. kasi yung unang nag ppt yung others. 2 main proteins present in egg white:
globulins and albumins. at varying [salt]s iba ibang proteins nagp-ppt out. some are more insoluble in dilute salt soln, some are more insol in
concentrated salt soln. technique used is salting in and salting outttttttttt
salting in:
iin dilute solns, soluble / do not want to ppt [assumption] salting in, papasok ng solution: you MAKE IT soluble. salting out:
nilalabas yung protein, only when conc salt []. in general. there are exceptions, though.
assume that globulins ppt out first [ explanation]
same [salt] added. pero bakit hindi same time nag ppt out? saturated yun
salting in: trying to dissolve in to the protein ewan
casein: heterogenous mixture of proteins found in milk, phosphoprotein produced in the mammary tissue; nutritionally-adequate protein bc essential aa’s are present in it; pI 4.7
main > isoelectric pption. also : lowering of dielectric constant of H2O. oks na pag ~pH 4,wash w ethanol > loweing o diel const . acetone more volatile.
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isoelectric focusing is like SDS-PAGE: apply voltage based on protein’s pI. based on paper
presentation of data: yield of albumin, do not compare it to the casein. statistical coeff: normalizeeeee (recording)
extr isol purifxn.
isol :albumin – salting out casen – isoelectric pptation
E2 PROTEIN QUANTITATION THROUGH SPECTROPHOTOMETRIC METHODS REaCTION W SUBSTRATESM ETCE TEC TECTERCETRCTE
confirm if may proteins nga na nakuha.
spectrophotome
tric: Use absorption of cpd of light; absorbance is directly prop to concentration: Beer-Lambert’s Law. A = epsilon * b *c
Applicable only when compound is capable of absorbing light in the UV-VIS region
Properties of
proteins The diversity of protein structure must be considered in choosing the most suitable protein quantitation method. - GLOBULAR PROTEINS
- eg albumin, antibodies. - soluble in dilute salt soln - FIBROUS PROTEIN - collagen, keratin
- typically insoluble (in salt solns) unless hydrolyzed - CONJUGATED PROTEINS
- Hemoglobin. proteins w non-aa components eg glycoprotein, lipoprotein. prosthetic group. (iron ligand = heme)
Casein is conjugated because it has phosphate group. Basis for
quantitative measurements
PHYSICAL PROPS
- presence of aromatic aa’s w/c absorbs EM rad maximally at 280 nm
CHEMICAL PROPS (react to make PRODUCTS which absorb…)
Brilliant Blue]
- peptide bond forming violet-colored complex with Cu2+ [Biuret]
- reaction with oxidizing agents of Tyr and Trp to phosphotungstic and phosphomolybdic acids [BCA?]
ASSAYS IN THE
EXPT BRADFORD ASSAY- Bradford reagent (Coomassie Brilliant Blue G250 in 95% ethanol and 85% H3PO4)
- standard calib curve is constructed using BSA as the std protein
like typical uv-vis. std calib curve, then conduct analysis of sample. have known, fit in unknown.
(absorbance shift 465 nm 595 nm) (read at 595) ADVANTAGES: simple, sensitive (up to 5 micro g / micro L), few interferences
Interference: siya mismo mag aabsorb, or
aagawan yung complex to disassemble complex ganern
Disadv: formation of dye-protein complex can be affected by the number of basic amino acids.
Reading changes in basic aa’s. false result pag super basic na kasi di kakabit coomassie blue. BIURET ASSAY
- Biuret reagent: NaOH, hydrated copper (II) sulfate - rxn bet Cu2+ in alkaline soln and 2 adjacent
peptide bonds
pinakamahalaga ang Cu2+. it binds to peptide bonds.
peptide bonds chelated by Cu2+, which is then reduced to Cu+
@540 nm
Cu2+ Cu+. reduced. cannot retain +2ness.
ADVANTAGE : not affected by AAcomposition; detects peptide bond easily
DISADV: lacks sensitivity. cannot read when less than 1 mg/mL ang sample.
Other
quantitation methods
Lowry
- combo of Biuret assay and [O] of tyr and trp w Folin and Ciocalteau’s reagent
- more sensitive than Biuret Bicinchonic
- similar to Biuret, chelation of bicinchoninic acid to Cu+
- first, proteins reduce Cu2+ to +. bicinchoninic kakabit sa Cu+.
- most sensitive assay :’) Ninhydrin
- det’s free amino nitrogen
- ninhydrin is yellow in color, reacts with N producing deep purple soln
kung may free amino groups, dun kakabit yung
ninhydrin: Lys, Arg (guanidinium grp: N=N-N), terminal NH3+. these are susceptible to ninhydrin rxn.
Kjeldahl
- measures total nitrogen in a sample
very common. used in food companies to det prot contents. melamine scandal!—reported as high prot when really just high melamine (wc is N rich)
cleave N bonds. protein gagawing ammonium ion ammonia. then titrate to get %N.
disadvantage: attributes all N% to the amino group, but not the side chain. not accurate. small
discrepancy usually
diff proteins have diff hydrophobic regions….. how do you know if that’s really the protein? ganern.
standard. to correct for side chains