EXPERIMENT 1: Proteins -polypeptides compose of > 50 amino acids Functions 1. Enzymes or subunits of enzymes-enhancing the rates of reactions 2. Structural or Mechanical roles 3. Immune response roles 4. Storage and transport of substances 5. Source of amino acids for organisms that cannot synthesize amino acids naturally ISOLATION- disruption of cell membranes to release cell contents; separation for other contaminants 1. CENTRIFUGATION 2.
SALTING OUT-water is made available to the protein by adding a salt solution of higher concentration 3. ISOELECTRIC PRECIPITATION- varying the pH conditions to reach protein pI * pI-pH at which the net charge of all its associate groups is equal * protein aggregates since the electrostatic repulsive forces are at a minimum. 4. Precipitation with organic solvents or acidic protein precipitants -addition of O. S decreases dielectric constant
PURiFICATION 1. Column chromatography 2. Density gradient centrifugation 3. Electrophoresis 4. enzymatic digestion 5. Removal of small ions or molecules or to exchange one ionic environment for another during purification is done by :Dialysis, Ultrafiltration and Gel filtration Dialysis-addition of NaOH and HClm PROTEIN STRUCTURES 1. Fibrous –not water soluble: Collagen 2. Globular- mostly water soluble : Globulin, Hemoglobin and Albumin-Globular A.
Invertase- catalytic protein or enzyme that catalyzes the breakdown of sucrose sugar- mixture of fructose and glucose or inverted sugar syrup -industries and Invertase is usually denied from yeast; may also be synthesized from bees -Optimum temperature at which the catalyze rate is at its greatest is 60 o and with optimum pH of 4. 5 – Sucrose -> Invertase-> glucose + fructose B. Casein- globular proteins; tend to fold back on themselves into compact, nearly spherical units, relatively hydrophobic but easily solubilized in to as colloidal suspension. with numerous of pro-residues that do not interact, no disulfide bridges -relatively little 3o structure -phosphoprotein(PO43-) attached to OH- groups of some amino acid R-groups acts as food source and supplies amino acids and inorganic elements CA and P. -pI – 4. 6 pH ; insoluble at less than 4. 6 -pH= 6. 6 – casein has (-) charge in milk and is solubilized at salt, exists in milk as Calcium caseinate -if acid is added, (-) charges on the outer surface micelles are neutralized by protonation at the phosphate groups and the neutral protein precipitates with the Ca 2+ ion remains in solution casein is precipitated by simply adjusting the pH of milk to be sufficiently acidic that the isolated casein is insoluble in water, alcohol and ether, both dissolves in alkaline and some acidic solution. – a nutrient protein has energy that can be realeased during metabolism – cleavage of unstable phosphate group release the energy C. Albumin – protein that is soluble in water and also in moderately conc.
Salt solutions D. major plasma protein, maintain plasma colloidal osmotic Pressure contributing to the regulation of osmosis, helps to transport hormones, drugs and other substances through the blood -transport protein for large organic anions in some hormones when their specific binding are saturated -human body: transports essential fatty acids for adipose tissue to muscle tissues -ANTIOXIDANT- used to treat people with certain types of poisoning 4 amino acids PRECAUTIONARY ROLES 1. Control of Temperature 2. Tempreature should be as low as possible to minimize thermal denaturation and the activity of the proteolytic enzyme 3. H must be mainained to prevent denaturation of due to fluctuation in pH Principles 1. Invertase- Precipitation with organic Solution ang addition of 95 % ethanol 2. Albumin- Salting out with Ammonium Sulfate 3. Casein- pI with HCl 4. Globulin- dialysis Methods of extraction 1. Repeated freezing and heating 2. Homogenization at high pH 3. Filtration 4. Sonication Defat- remove lipids and other substances to purify Grind- break cell wall BRADFROD ASSAY -binding of coomasie brilliant blue dye to the protein under acidic conditions causes shifting of wavelength from 465 to 595 nm -can be used to determine the protein conc.
In the range of 1-20 microgram/ml -unaffected by the presence of most non-protein components of cells biological extracts WARBURG-CHRISTIAN METHOD – % NA= 100 5- A280/260 -Protein concentration (mg/mL) = 1. 55 A280 – 0. 76 A260 ENZYMATIC CATALYSIS CATALASE -prevents accumulation of toxix leves of h202 formed as a byproduct of metabolic processes specifically ETC -has 4 polypeptide chain, each composed of more than 500 A. A -HEME group responsible for the catalytic enzyme activity