Lets build a spectrophotometer Light source sample 1

Let’s build a spectrophotometer Light source sample 1) Measure Io (100% T) 2) Measure I (T of sample) 3) Calculate A detector

Scanning an absorbance spectrum Light source sample monochromator detector A Change the monchromator to measure A versus wavelength Resolution will be defined by dispersion and slit width, bandpass ≤ 2 nm is sufficient λ (nm)

Measure all wavelengths at once Light source sample Detector array monochromator Diode array spectrometer 1) Sample receives broad irradiation (i. e. , white light) 2) Dispersion element comes after the sample 3) Project spectrum onto an array of detectors

Some applications of spectrophotometry 1) Concentration Proteins DNA 2) Environmental effects Effect of solvent p. H Ligand or protein interactions 3) Time – resolved measurements Measure time dependent changes Timescales

Measuring concentration Suppose we are studying an enzyme that consumes NADH as part of its catalytic reaction, we can use the long wavelength absorbance band of the pyridine chromophore to measure NADH concentration, and thereby activity. A + H+ + NADH → NAD+ + AH 2 We know the KM of the enzyme for NADH is 100 μM (and for A, KM =5μM) We wish to measure the total activity present in a series of extracts. Measure Vmax at saturating S, (i. e. , NADH & A) [NADH] = 1 m. M (10 -fold> KM) Measure A at 340 nm, A = ε c l A= 6. 23 x 103 M-1 cm-1 (1 x 10 -3 M) = 6. 23

Measuring concentration (contd) A > 6 means % T < 0. 0001 % , 99. 9999 % of Io absorbed Most spectrophotometers are only linear up to A= 3 Stray light How can we fix the problem? A 3 Dilute the NADH. 2 What about the pathlength? 1 Measure off the peak. [x] (M)

The spectrum of NADH 2. 0 20 NAD+ ε (m. M-1 cm-1) A 1. 0 NADH 0 10 0 220 300 400 Wavelength (nm)

Environmental effects-Solvent perturbation a) Trp in aqueous buffer 2. 0 A b) + co-solvent e. g. , 10% DMSO 1. 0 E 2 Red- shift Longer λ 0 260 280 Wavelength (nm) 300 E 1 Blue-shift to Shorter λ

Difference spectroscopy- small changes ΔA Using trp in aqueous buffer as reference, i. e. , 100 % T 0. 1 0. 05 0 -0. 05 -0. 1 260 280 Wavelength (nm) 300

Split-beam spectrophotometer mirror Reference Trp in buffer Beam splitter Sample Light source Trp + DMSO monochromator detectors

Split- beam or split cuvette In the two compartments (1 and 2)we have two proteins A and B that we suppose form a complex, A+B AB Place equal volumes of the two protein solutions in the two sides, Measure this as reference, i. e. , 100 %T, then mix and record again, The difference spectrum is generated 1 2 Light source sample monochromator detector

Ligand binding Cyanide binding to the respiratory enzyme- cytochrome c oxidase What we know, cyanide is a potent poison of respiration CN inhibition to respiring mitochondria is instantaneous Blocks electron transfer to O 2 S [O 2] M CN Time (min)

Cyanide reaction with cytochrome c oxidase A ACN=εCNcl 1 A=εcl 0. 5 A 430 nm E-CN E +CN 0 400 420 440 460 Wavelength (nm) 0 1 10 100 Time 1000

Time-resolved spectroscopy Time (m) 1000 ΔA 1) Isosbestic points 700 0. 4 400 0. 2 2) E + CN 100 1 0 E-CN 3) Reactivity of the enzyme in vitro is different from enzyme in vivo 4) Oxidase exists in multiple states -0. 2 -0. 4 400 420 440 Wavelength (nm) 460