逻辑门电路基础CreateTime

1、逻辑门电路基础CreateTime“A basic comprehension of the methods described here is necessary for an appreciation of the significance and the limitations of the information presented in the text”逻辑门电路基础CreateTimeProtein IsolationMust have sensitive method for detection.Select a good source for the protein.a. R

2、ich source of material.i.e. Heart mitochondria for cytochrome C b. bakers yeast (Saccharomyces cerevisiae)c. Escherichia coli (recombinant expression)Tissue specificity: Brain vs. kidney vs. eye.Chickens, cows, pigs or rats are often used.Molecular cloning techniques have allowed biochemists to over

3、-express desired proteins in bacteria or C.H.O. (Chinese Hamster Ovary) cells by isolating the gene and placing it into a host system.逻辑门电路基础CreateTimeMethods of solubilization animal cellsCells can be lysed by hypotonic shock.Cells with high salt inside and no salt outside willswell and ruptureBact

4、eria outer membranes must be digested.Gram-negative bacteriaHen egg white lysozyme digests b (1-4) linkages in the (glycosidic bonds) of polysaccharides.Mechanical breakage blenders homogenizersFrench press - high pressure 20,000 lbs/in2 forced through a small hole disrupts cells ultrasound or sonic

5、ation disrupts cells.逻辑门电路基础CreateTimeCentrifugationLysate - broken (lysed) cells- can be separated usingdifferential centrifugation RPM - “spun down”separates by density differences or by size (MW) of particles.Cellular fractionation can separate:mitochondriamicrosomesribosomessoluble proteins逻辑门电路

6、基础CreateTimeCentrifugation: UnitsWhere:w = angular velocityv = velocity of particleR = distance from center of rotationM = molecular weightV = partial specific volume of particler = density of solventSedimentation velocity (Svedberg Coefficient) S = s x 10-13逻辑门电路基础CreateTimeH-bonds, ionic bonds, Va

7、n der Waals interactions, and Hydrophobic interactions can be disrupted. Denaturation is the process by which a protein loses its “native” or active shape or conformation.Temperature can play a role“cold labile”“heat labile”Protect against-Proteases, Inhibitors, Changes in pH,Protein can be air-dena

8、tured -egg white meringue - absorption to surfaces Damaged by oxidation 02Heavy and transition metals damage proteins -they bind to protein- Cu+ Hg+Bacterial contamination can destroy the proteinStability: proteins can denature!逻辑门电路基础CreateTimeIn order to follow the purity of an enzyme, you need a

9、method to measure its activity.Spectraphotometric analysis- is one common method to measure activity.Substrate S Product P a change of S with timeif S is colored “absorbs light” we can use Beers Law.A = eb c c - concentratione - millimolar extinction coefficientA - absorbance b - path lengthT - perc

10、ent transmittanceActivity MeasurementsA = - log % Tif A then c at max逻辑门电路基础CreateTimeFor the reaction: NADH NAD+ + H-enzymeAbsorbance300 nm350 nmNADHNAD+DAl Max = 340 nmDT minmgmg of protein= Specific activityVolume is 1 ml so micromoles NADH oxidized逻辑门电路基础CreateTimeStart with one liter of lysed c

11、ells.We measure the rate of .01 ml of cells at at concentration of 20 mg/ml. i.e. the amount of enzyme we will assay is 0.01 mlWe get a rate of A = 0.5 A/min1 millimolar = 6.22 DA = e mM1 millimolar in a volume of one ml = 1 micromole/ml = moleC=.008 moles in 1 ml/min = .04 moles 0.2 mg min/mg逻辑门电路基

12、础CreateTimeTotal activity: .04 mmoles x 20 mg/ml = 0.8 mmoles / ml moles x 1000 ml = 800 moles in 1 liter of cells ml minRed = is our enzymeIf we remove greens & blues the specific activity increases, however, our total activity remains the same.If We lose red the total activity decreases.逻辑门电路基础Cre

13、ateTimeWe usually monitor both the total activity and specific activity for each purification step.Until the Specific Activity reaches a maximal value.How do we know if it is pure? Usually SDS - PageSee Table 5-4 in Voet and VoetSome enzymes have no easy assay but the product of the reaction can be

14、used in another reaction: enz1 enz2A B C NADH NAD+Coupled Reactions: We couple enz2 to enz1 and measure NADH to get A逻辑门电路基础CreateTimeUse of radioactivityATP ADP + PiSeparate ATP + Pi + ADP on TLC measure radioactivityPhosphoimager makes this easy else cut spots and count in scintillation counter.Pi

15、ATP逻辑门电路基础CreateTimeStrategy of PurificationFractionation procedures or steps to isolate protein based on physical characteristics.Characteristic ProcedureCharge 1. Ion exchange2. Electrophoresis3. Isoelectric focusingPolarity 1. Adsorption chromatography2. Paper chromatography3. Reverse phase chrom

16、atography4. Hydrophobic interaction逻辑门电路基础CreateTimeCharacteristic ProcedureSize1. Dialysis and ultrafiltration2. Gel electrophoresis3. Gel filtration4. UltracentrifugationSpecificity1. Affinity chromatography2. ImmunopurificationSolubility1. Salt precipitation2. Detergent solubilization逻辑门电路基础Creat

17、eTimeIonic StrengthCi = the molar concentration of the ith speciesZi = its ionic charge1M Na+ Cl- Z = 1 Na+Z = 1 Cl- 1 = (1M x 1)Na + (1M x 1)Cl 2逻辑门电路基础CreateTimeFor di- or tri-valent ions, where I is different than M1M MgCl2 Mg+ = 1M, and Z = 2whileCl- = 2M, and Z =1I = (1 x 22)Mg + (2 x 12)Cl = 4

18、 + 2 = 3 2 2逻辑门电路基础CreateTimeSalting outUse (NH4)2 SO4 : it is a Very Soluble salt that does not harm proteins.Refer to the Hofmiester Series逻辑门电路基础CreateTimeSolubility of carboxy-hemoglobin at its isoelectric point逻辑门电路基础CreateTimeSolubility of b-lactoglobulin as a function of pH逻辑门电路基础CreateTimeCh

19、romatographyAnalytical methods used to separate molecules. Involves a mobile and a stationary phase.Mobile phase is what the material to be separated is dissolved in. Stationary phase is a porous solid matrix which the mobile phase surrounds. Separation occurs because of the differing chemistries ea

20、ch molecule has with both the mobile and stationary phase.Chemistries are different depending on the specific method.逻辑门电路基础CreateTimeTypes of chromatographyGas - Solid: Mobile phase is gaseous, stationary phase is a solid matrix.Liquid - Solid: Mobile phase is liquid, stationary phase is a solid ma

21、trix. If separation is based on ionic interaction the method is called Ion Exchange chromatography.If separation is based on solubility differences between the phases the method is called adsorption chromatography. If the separation is base on size of molecule the method is called gel filtration or

22、size exclusion.If the separation is base on ligand affinity the method is called Affinity chromatography. 逻辑门电路基础CreateTimeIon Exchange ChromatographyA solid matrix with a positive charge i.e. R+ can bind different anions with different affinities. We can s counter ion for another(R+A-) + B- (R+B-)

23、+ A-R = Resin and exchanges Anions (-)This is an anion exchange resin.There are also cation exchange resins. The type of an R group can determine the strength of interaction between the matrix, R and the counter ion.If R is R-(R-A+) + B+ (R-B+) + A-逻辑门电路基础CreateTimeProteins have a net charge.The cha

24、rge is positive below pI,while the charge is negative above pIThe choice of exchange resin depends on the charge of the protein and the pH at which you want to do the purification.Once the protein binds, all unbound proteins are washed off the column. Bound proteins are eluted by increasing the ioni

25、c strength, changing the counter ion or changing the pH altering the charge on the protein or the column. 逻辑门电路基础CreateTimePaper chromatographyStationary phase vs. the Mobile phasePartitioning between the two phasesPartition coefficientThe more H2O soluble the slower it migrates.The more organic sol

26、uble the more it migrates.The aqueous component of the solvent combines with the cellulose of the paper and becomes the stationary phase.逻辑门电路基础CreateTimeMaterials can be visualized by:RadioactivityFluorescenceUV absorbencyStained with one of several dyesNinhydrinIodineSulfuric acid逻辑门电路基础CreateTime

27、Ninhydrin visualizes amino acids逻辑门电路基础CreateTimeTwo dimensional separation of Amino acids逻辑门电路基础CreateTimeGel FiltrationSize exclusionA matrix with holes in it.Vt = Vx + VoVo = void volume = volume outside the “caves or knooks and crannies”Vx occupied by gel beadsVo 35% of Vt逻辑门电路基础CreateTimeVe = e

28、lution volume Vo = exclusion volumeCommon matrix: dextran, agarose, or polyacrylamidealso desalts proteinsGel filtration can be used to determine the molecular mass of proteins逻辑门电路基础CreateTimeBefore swelling the dry bead size 5% of Vt 60% are “holes”Hole sizes can be made differentSmall molecules s

29、ee a larger column volume than big molecules and they get hung up in the caves. Large proteins are excluded, while small protein are included.Separation on size and shape.逻辑门电路基础CreateTime逻辑门电路基础CreateTimeDialysis is a process that separates molecules according to size through the use of semipermeab

30、le membranes containing pores of less than macromolecular dimensions逻辑门电路基础CreateTimeAffinity ChromatographyBased on molecular complementary between an enzyme and substrate.The substrate (R) is linked to a matrix with a spacer armOnly protein that binds R will stick to column. put citrate on column citrate dehydrogenase will specifically bind. Add excess citrate and the enzyme will be released.逻辑门电路基础CreateTimeThe purification of Staphylococcal nuclease using the ligand, diphosphothymadine逻辑门电路基础CreateTimeElectrophoresisThe migration of ions in an electric fieldFele = qE where q is the char