Protein detergent interactions - Nano Science experimetal excercise

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1 April 25, 2006 Protei deterget iteractios - Nao Sciece experimetal excercise Ja Skov Pederse, Bete Olse ad Petra Bäverbäck Departmet of Chemistry ad inano Iterdicipliary Naosciece Cetre, Uiversity of Aarhus, DK-8000 Aarhus C, Demark jsp@chem.au.dk Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, or SDS-PAGE, is a techique used i biochemistry ad molecular biology to separate proteis accordig to their size (legth of polypeptide chai). I this exercise we will ivestigate the protei-deterget complexes formed whe the samples are prepared for the techique. The proteis are deaturated by the aioic deterget sodium dodecyl suphate (SDS): SDS has a polar head group which is hydrophilic (likes water) ad a hydrocarbo chai which is hydrophobic (hates water). Above room temperature the Na io dissociates from the head group ad SDS is dissolvable i water. At low cocetratio it is dissolved as sigle molecules despite the ufavorable iteractios betwee tail ad water. This is due to the large etropy of mixig at low cocetratio, which overcomes the ufavorable cotributios. At higher cocetratios, the molecules aggregate to reduce the ufavorable cotact with water, ad small globular micelles are formed which are surrouded by the polar head groups. The cocetratio where the micelles start to form is called the critical micellar cocetratio (CMC). Idividually dissolved molecules are also preset above CMC with a cocetratio of about CMC as these molecules have large etropy of mixig. Whe mixed with proteis, SDS works by disruptig o-covalet bods i the proteis, thereby deaturig them, causig the molecules to lose their ative shape (coformatio). Also, aios of SDS bid to the mai peptide chai. This effectively imparts a egative charge o the protei. This ew egative charge is sigificatly greater tha the origial charge of that protei. The electrostatic repulsio that is created by bidig of SDS causes proteis to ufold ito a rod-like shape thereby elimiatig differeces i shape as a factor for separatio i the gel. Besides the additio of SDS, proteis are ofte prepared i the presece of a reducig aget, such as dithiothreitol (DTT) or mercaptoethaol, which further deatures the proteis by reducig disulfide likages, thus overcomig some forms of tertiary protei foldig, ad breakig up quaterary protei structure (oligomeric subuits). This is kow as reducig SDS-PAGE, ad is most commoly used.

I this exercise beta-mercaptoethaol: is used for breakig the disulfide bridges of the protei. The bidig of SDS to the protei chai will give a egative charge to each protei i proportio to its mass.

The SDS bids to the protei i a ratio which is early idepedet of the protei primary structure ad this gives a approximately uiform mass:charge ratio for most proteis, so that the distace of migratio

2 I this exercise beta-mercaptoethaol: is used for breakig the disulfide bridges of the protei. The bidig of SDS to the protei chai will give a egative charge to each protei i proportio to its mass. For the gel electrophoresis, a voltage is applied to the gel ad this makes the proteis migrate. The SDS bids to the protei i a ratio which is early idepedet of the protei primary structure ad this gives a approximately uiform mass:charge ratio for most proteis, so that the distace of migratio through the gel ca be assumed to be directly related to oly the size of the protei. A trackig dye may be added to the protei solutio to allow the experimeter to track the progress of the protei solutio through the gel durig the electrophoretic ru. The electrophoretic cell, a example of a gel ad of a calibratio curve: The trace at the left ad right -had side is stadards, which are used for calibratig the masses. We study the protei BSA (bovie serum albumi, which is supplied as a powder). BSA is the most abudat protei i plasma with a typical cocetratio of 5 g /00 ml. 2

M = 66 000, 580 amio acids residues. Whe dissolved i water at a cocetratio of about 2wt%, the solutio attais a ph of about 7 ad BSA is slightly egatively charged.

Usig various mixtures of BSA ad SDS ad measurig the coductivety of the solutio, we will determie the amout of SDS which associates with BSA i solutio.

We also mix BSA with a oppositely charged deterget, dodecyl trimethyl amoium bromid (DTAB): M = 308.

3 M = , 580 amio acids residues. Whe dissolved i water at a cocetratio of about 2wt%, the solutio attais a ph of about 7 ad BSA is slightly egatively charged. I this exercise we study the iteractio betwee BSA ad deterget. Usig various mixtures of BSA ad SDS ad measurig the coductivety of the solutio, we will determie the amout of SDS which associates with BSA i solutio. Sice BSA ad SDS are both egatively charged, the iteractio betwee them is maily of hydrophobic ature. We also mix BSA with a oppositely charged deterget, dodecyl trimethyl amoium bromid (DTAB): M = g/mol DATB forms micelles i solutio at room temperature, ad measuremets have show that 25% of the Br ios are dissociated. Sice BSA is egatively charged ad DTAB is positively charged, they will associate. The DTAB micelles will electrostatically bid to the BSA molecules ad as they ca iteract with more tha oe BSA molecule, they will lik them together so that macroscopic aggregates are formed at charge eutralizatio. This will give rise to to turbidity of samples. Whe addig more DTAB, the electrostatic repulsio betwee the micelle-protei complexes become so large that the aggregates dissolve agai ad are charge stabilized. 3

Whe two electrodes i the solutio are made part of a complete electrical circuit, the catios (+) are attracted to the egative pole (cathode) ad the aios (-) are attracted to the positive pole (aode).

4 Coductivety of solutios Whe the compoud AB is dissolved i water, ios A + ad B - are produced. The coductivity of ioic solutios is the result of the movemet of ios through the solutio to the electrodes. Whe two electrodes i the solutio are made part of a complete electrical circuit, the catios (+) are attracted to the egative pole (cathode) ad the aios (-) are attracted to the positive pole (aode). Chages i the coductivity of a electrolyte solutio may result from chages i both the umber ad the mobility of the ios preset. Both aios ad catios cotribute to the overall coductivity. I the solutios we will i geeral have sigly dissolved SDS molecules/ios ad their couter ios, SDS micelles ad their couter ios, BSA, BSA-SDS complexes ad couter ios. All of these species have differet charges ad differet sizes ad therefore also differet mobilities. Whe SDS is dissolved as sigle molecules, the couterios are fully dissociated. I the micelles ad complexes with micelle-like aggregates, the couterios are ot fully dissociated ad this gives a much smaller effective charge per SDS molecule i the micelle compared to those dissolved idividually. At the low cocetratios used i this exercise, the measured coductivity ca be cosidered to be the sum of the coductivity of the various species. We will ivestigate the coductivity of a pure SDS solutio as a fuctio of cocetratio ad use this for providig a rough estimate of CMC of SDS. Due to the high mobility of the idividually dissolved SDS molecules ad their couter ios, the coductivity icreases rapidly with cocetratio below CMC. The icrease i coductivity above CMC is slower due to the lower degree of dissociatio of the SDS micelles. For the SDS-BSA mixtures, we will have a costat cocetratio of BSA ad icrease the cocetratio of SDS. For low cocetratio of SDS, the idividually dissolved SDS molecules ad the couter ios provide agai the largest cotributio to the coductivity. As SDS associates with BSA, the dissociatio of the added SDS goes dow ad the coductivity icreases slower with cocetratio. Whe the BSA is saturated with SDS, free SDS micelles are formed. These have a differet dissociatio tha the SDS i the SDS-BSA complexes ad therefore the coductivity chages agai. From the variatios i coductivity we ca estimate the amout of SDS boud to the BSA. 4

Turbidity measuremets There are several practical ways of quatifyig cloudiess of solutios, the most direct beig some measure of atteuatio of light as it passes through the sample.

5 Turbidity measuremets There are several practical ways of quatifyig cloudiess of solutios, the most direct beig some measure of atteuatio of light as it passes through the sample. This atteuatio ca be measured usig a spectrophotometer. Pricipally it is doe as: The atteuatio of light at a wavelegth λ by a solutio is described by the equatio I( sample) / I( o sample) = exp( τ d) where τ is the turbidity ad d is the sample thickess. Notice that the photometer gives absorptio [ I( sample) / I( o sample ] = τ d A = l ) We will prepare a series of mixture of BSA ad DTAB with icreasig amouts of DTAB ad costat cocetratio of BSA ad measure the turbidity of the mixtures. The associatio of the DTAB micelles ad BSA is strogest whe the positive charge of the DTAB equals the egative charge of the BSA, sice the formed aggregates are charge eutral. Due to the bridgig by the DTAB micelles, the turbidity is also largest at charge eutrality. Usig the kowledge that the dissociatio of the couter ios of DTAB is 25%, we ca use this for calculatig the charge of BSA. Experimetal procedures Samples To make the samples (stock solutios) we will eed: 2 wt % Bovie Serum Albumie (BSA) i 50 mm β-mercaptoethaol: 50 ml. 6 wt % Sodium Dodecyl Sulfate (SDS): 50 ml. wt % Dodecyl Trimethyl Amoium Bromide (DTAB): 25 ml Deioized water First weigh out the amout of protei ad surfactats eeded for each solutio ad the add the water. Mix carefully util the powders have dissolved completely. Avoid to shake the samples too 5

6 much so that excessive foam is created. The stock solutios should rest for at least 20 mi before you start further mixig followig the tables below. After that, add the β-mercaptoethaol to the BSA solutio. Hadle the β-mercaptoethaol i a fume cupboard as it is poisoous ad smells bad! Safety data sheets for all chemicals used are available i the lab ad at Make three sample series: oe with BSA ad SDS, oe with oly SDS ad oe with BSA ad DTAB. Follow the mixig schemes i tables -3 whe you make the samples. Table. Mixig scheme for the BSA + SDS series. Sample No. Volume BSA (ml) Volume SDS (ml) Volume H 2 O (ml) b b b b b b6 4 3 b b b b b b b b b b6 4 3 b b b b Coductivity 6

7 Table 2. Mixig scheme for the SDS series. Sample No. Volume SDS (ml) Volume H 2 O (ml) s 0 8 s s s s s6 7 s s s s s 2 6 s s s s s6 3 5 s s Coductivity Table 3. Mixig scheme for the DTAB series Sample No. Volume BSA (ml) Volume DTAB (ml) Volume H 2 O (ml) d d d d d d d d d d d 2 d d d d d d Coductivity 7

8 Coductivity measuremets. Calibrate the electrode. You will fid the istructios beside the apparatus. 2. Measure the coductivity of the samples with both BSA ad SDS (the b-series) ad with oly SDS (the s-series), followig the istructios below. a. Put the sample i the measurig cylider ad the electrode i the liquid. Remember to check that the four metal stripes are covered by liquid. b. Whe the display says STAB, read ad write dow the coductivity. c. Save the sample i its tube. d. Use a piece of tissue to clea the cylider ad the electrode before measurig the ext sample. Absorbace measuremets Measure each BSA + DTAB sample (the d-series) as quickly as possible after makig them, ad the save them i their tubes. Istructios are foud beside the apparatus. Use a wavelegth of 400 m. Data treatmet ad aalysis Plot usig Excell the coductivity of the SDS cocetratio series as a fuctio of SDS cocetratio. Provide ad estimate of the CMC of SDS i uits mm. Plot usig Excell the coductivity of the SDS-BSA mixtures as a fuctio of SDS cocetratio. I order to locate the poit where the coductivity starts icrease agai due to the formatio of free SDS micelles, we calculate the slope of the measured coductivity curve. For the measuremets (x,y ) with =, N, we calculate the slope at x as the average slope betwee x - ad x : y x y x ad betwee x ad x + y x + + y x. For equidistat poits with the separatio Δx it reduces to: y+ y α = which is easily calculated ad plotted with Excell. 2Δx The poit just before the icrease i α provides the maximum amout of SDS associated with BSA. Calculate how may grams of SDS associates with oe gram of BSA. How may SDS molecules per amio acid residue does this correspod to? The desitete of BSA i solutio is approximately.35 g/cm 3. Assumig that the molecule is spherical, what is the radius? 8

9 I the ufolded state, we cosider BSA to be oe log liear molecule with oe C-C ad two N-C bouds per amio acid residue alog the backboe. This correspods to a legth of m alog the backboe. What is the legth of the liear chai? What is the radius of the liear chai if we assume that it is a straight cylider? SDS has a desity of about.05 g/cm 3 i solutio. Usig the amout of SDS determied from the coductivity measuremets, what is the total volume of SDS associated with oe BSA molecule? We assume that SDS makes a cylidrical shell aroud the ufolded liear BSA molecule. If we assume that the thickess of the shell correspods to the legth of the C2 chai of SDS of l =.68 m, what is the legth of the SDS shell ad what fractio of the total BSA legth does it correspod to? How do you thik the SDS-BSA complex look? For the DTAB-BSA mixtures, use the results from the maximum i turbidity for calculatig the charge of BSA, assumig that the degree of dissociatio is 25% for DTAB. 9

CHAPTER 11. Practice Questions (a) OH (b) I. (h) NH 3 CH 3 CO 2 (j) C 6 H 5 O - (k) (CH 3 ) 3 N conjugate pair

CHAPTER 11. Practice Questions (a) OH (b) I. (h) NH 3 CH 3 CO 2 (j) C 6 H 5 O - (k) (CH 3 ) 3 N conjugate pair CAPTER 11 Practice Questios 11.1 (a) O (b) I (c) NO 2 (d) 2 PO 4 (e) 2 PO 4 (f) 3 PO 4 (g) SO 4 (h) N 3 (i) C 3 CO 2 (j) C 6 5 O - (k) (C 3 ) 3 N 11.3 cojugate pair PO 3 4 (aq) C 3 COO(aq) PO 2 4 (aq)