Measuring Protein Activity (Spectroscopy) PDF

Measuring protein activity (spectroscopy) Cellular Biochemistry and Biology Dr Bunmi Ibrahim  Learning outcomes light ~ 1. Explain the principle of spectroscopy 2. Describe how spectroscopy can be used to measure multiple...

Measuring protein activity (spectroscopy) Cellular Biochemistry and Biology Dr Bunmi Ibrahim  Learning outcomes light ~ 1. Explain the principle of spectroscopy 2. Describe how spectroscopy can be used to measure multiple molecules 3. Use the Planck-Einstein equation to explain the relationship between wavelength and energy 4. Discuss factors that can influence the protein absorption spectra 5. Use the Beer-Lambert equation to explain the relationship between absorbance and concentration 6. Describe the effect of stoichiometry in the use of spectroscopy What is spectroscopy? + Emission Absorption - an occur ↓ Light and matter always interact with each other in a specific way. Spectroscopy is therefore the study of the absorption and emission of electromagnetic radiation (light) by matter. Loading… A beam of light on a ‘prism’ can make the light separate into different components (a spectrum). Some of these components are visible while others are not. 2 def  Why measure proteins? Spectroscopy is useful for answering questions such as: location 1. Where is the protein? (bound or free) identification 2. Which protein is present? (size/charge) quantification 3. How much of the protein are there? (concentration) the effectivement activity of neprotein 4. How effective is the protein? (activity) Electromagnetic radiation (EM) The energy carried by EM depends on wavelength and frequency. Low wavelength will give high energy E= Loading… hc λ E = energy, h= Planck’s constant, c= speed of light, λ= wavelength Quiz Ultraviolet-Visible spectroscopy Mainly used in Biology UV-Vis radiation is high energy. It excites delocalised electrons. Where will you find delocalised electrons?  Absorbance and spectroscopy σ* (anti- bonding) π* (anti-bonding) Atoms will absorb light at a specific, unique wavelength E n n (non-bonding) e π (bonding) r Electrons in the atom move from ground state to excited g state y σ (bonding) At a given wavelength, how much light is absorbed can be used to estimate the concentration of the sample The absorption spectrum gives a fingerprint of a biological molecule  Top has higher Example it absorbance , on nar A most a 'onugat Peptide bonds Aromatic residues = 280nm 200-230nm I Carboxylic moieties 2 Trp Ty rPhe or aromatic BS 1 A rings 0 200 250 300 350 night Wavelength (nm) lowe  Beer-Lambert relationship A = cl Absorbance (A ) is dependent upon: concentration (c) of solute light path length (l) molar absorption/extinction coefficient ( ) = absorbance of 1 M-1.cm-1 solution at wavelength ( ) of incident energy A is proportional to c  Example 0.000.010.020.030.040.05 C (mM) C (mM) A280 0 0 Loading… 0.01 0.18 Remember… A =. 0.02 0.37 cl 0.03 0.55 A = 0.04 0.72 c 0.05 0.91 l  What if there is no ‘colour’? Absorption spectra useful for identifying different chromophores & their concentrations. Nucleic Acid Wavelength and 1.6 Protein stoichiometry are 1.2 A Purity Ratios: b 260/280 = 1.8-2.0 s 0.8 260/230 >2.0 or b important a 0.4 n c e 0 220 240 260 280 300 320 340 Wavelength (nm) 1.2 t A 0.8 b s o 0.4 r b NAD a H n 0 NAD c 250 300 350 400 450 + 500 e Wavelength (nm) Example 1.2 Phosphoenolpyruvate A 0.8 b 340nm s o AD r 0.4 b NAD P P Problem – both ATP & a n H NAD 0 yr ADP absorb at 260nm c e 250 300 350 400 450 + Wavelength (nm) Mg2 u v At 340nm NADH absorbs but NAD+ does not + at As ATP is synthesised, NADH is oxidised e Absorption decreases over time AT K in NADH + NAD P as e H+ + Pyruvate Lactate Lactate Dehydrogenase NADH is visible by spectroscopy  Coupling reactions Stoichiometry is important A Slowe B B Faste C 1 r 2 2x( 1 r 2( 1 ↑ ne rate 2 4 limiting step Rate of reaction is important Substrate Product 1 Product 2 for even Here will be - 1 of A 4 of C Conformational/structural changes Protein absorption spectrum affected by microenvironment UV-Vis spectra of (a) fresh egg-white protein in deionised water (b) egg-white protein in gold nanoparticle colloidal solution and (c) post laser UV-visible spectra of the SPT wild type ( A ), N100Y ( B irradiated protein gold nanoparticle solution. ), and R378A ( C ) Joshi & Soni 2012. Raman et al., 2009 DO - 10.1074/jbc.M109.008680 DOI:10.1364/PHOTONICS.2012.MPo.3 Conformational changes due to ligand binding or mutations can be observed in the spectra Thermal denaturation ssDNA has a higher absorbance Heating induces transition from double stranded to single stranded DNA  The solvent effect The choice of solvent is important in the use of UV-Vis Some solvent interact with the solute molecules thus affecting the absorption peaks In proteins, some solvents can disrupt hydrophobic interactions in the side chains of the amino acid UV–Vis spectra of BSA in various buffers at different residues. concentrations (0.01, 0.05, 0.1 mol·dm⁻³) at 25 °C; a PBS, b TRIS and c imidazole buffer Satish et al., 2017. DO - 10.1007/s10953-017-0612-0  Summary Spectroscopy: used to measure how light (EM) interacts with matter (macromolecules). ?? The macromolecules have a specific wavelength and produces a spectrum (fingerprint). UV-Vis can determine the identity, concentration and activity of proteins. A= εcl (Beer-Lambert law) is important to calculate the concentration of substances. - When there is no colour, wavelength and stoichiometry are important for spectroscopy.  Title undert and relationship between energy ! Spectroscopy The energy carried by EM + wavelength & ↓ depends on 1 + frequency? def use ~ inverse relationship low 1 = high t What is wavelength UV spectroscopy high I low E = has high I sufficient mont uncu Planch - Eistein equation ! energy to excite/energise constant values are given (candh) ! delocated electrons ↓ in found in 1) aromatic moleules (benzene) 3- p[ due to 3 from 2) metalic ponding urlight the es go from ground - excited state example of exam a - the amount of light absorbed , at that specific wavelength, Why does a particular can be used to entimate cone of Protein/AA have night sample He des absorbance than peptide bonds 10 : 76-10 : 27 gap ↳ causes peak at around 220nm Stoichiometry Beer-Lambert 10 : 31 : EQ 10 : 30 10 23 on rely : Ar Exc : = the but wavdagm 11 colour T ↓ ↓ to furn Specific I me a in y 2) wavet light pan spectrum Absorbance length Can't see Colour molay Wen the is not a 11 : 3) coupling - reactions i 2 isdirectlryonal abortion stoichiometric ratio?. Ax it gusing con. e tomokale. to molas How well solute e.. g 1: 2 Conside page() absorb at ratios + same woming can a why is rate of reaction wavelength backward LavAz = low Conc (c) important ?. substant absorb light I given in exams  or mutation What affects the spectrum ? User iz structural - wavelength - can identify conformational the (microl environment changes in proteins missing - e g. - concentration absorption peaks - conformation/structure - 2)UV can dall Meter dsDNA has been ninally denaturated SsDNA has a higher absorbance man dsDNA Different solvents interact with the solute molecules affecting absorption peal + spectrum Exam Question : 10 : 46 e in Proteins. g. some sowent can disrupt hydrophobic interactions in R groups