BIOC0003 Term 1 - Lecture notes All term 1 lectures PDF

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BIOC0003 - Experimental Biochemistry Term 1 Lecture 1 pH, pK, buffers and equilibria pH is a measure of the amount of hydrogen ions (H+ ) in a solution pH = - log10[H+] What are the units of [H+]? As is usual with measurements of solution concentrations, the concentration of protons ([H+ ]) is measu...

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BIOC0003 - Experimental Biochemistry Term 1 Lecture 1 pH, pK, buffers and equilibria pH is a measure of the amount of hydrogen ions (H+ ) in a solution pH = - log10[H+] What are the units of [H+]? As is usual with measurements of solution concentrations, the concentration of protons ([H+ ]) is measured in units of molarity (M or mol.L-1 ). Why use - log10? Concentrations of protons in water can range over many orders of magnitude: from 1 M or more (very acidic) to 10-14 M or less (very alkaline). By taking the negative values of log10 of the Molarity, this converts to a more convenient scale Why is pH 7 “neutral”?

1. pH is temperature dependent (always use a temperature probe when you don’t work at ‘room temperature’) 2. The pH scale is logarithmic (a solution at pH 3.0 is not twice as acidic as a solution at pH 6.0, but 1,000 times as acidic i.e. it contains 1,000 times more H+ !) Acids and alkalis: An acid is a compound that dissociates in water to produce protons - the stronger the acid, the greater its tendency to dissociate. Likewise, an alkali is a compound that dissociates in water to produce hydroxide ions.

Why is pH important in Biology? Many enzymes & cellular activities are pH-dependent with an optimum pH for activity For example, the pH range of blood is very tightly controlled Dissociation of Weak Acids

^This is the Henderson-Hasselbalch equation - This is a centrally-important equation that can be used to: - determine the charge on a protonatable group at any pH - calculate amounts of reagents needed to make a buffer solution at a specific concentration and pH Titration of a weak acid, HA:

Many molecules have more than one titratable group and, therefore, more than one pK. - For example: Phosphoric acid is tribasic Consider the simple amino acid glycine. Both its amino and carboxylic acid groups are protonatable

The titration of Glycine:

pH, pK and buffers: If acid or alkali is added to mixtures of weak acids (HA) and their salts (A- ), the equilibrium position will shift to take up the H+ or OH- Hence, this decreases the changes in pH caused by acid/alkali additions - the mixture will buffer changes in pH

Titration of a weak acid:

Summary Points on pH Buffers: A weak acid can act as a buffer of pH because, at pH values close to its pKa : - it is a mixture of a weak acid (HA) and its salt (A- ); - it will minimise changes in pH caused by addition of acid or alkali by absorbing protons or hydroxides i.e. it will buffer the pH; - the proportions of acid and salt at a given pH, or the pH change caused by acid or alkali addition, can be calculated using the Henderson-Hasselbalch equation.

Measuring pH: - One way is with indicator dyes: these are weak acids in which the acid and conjugate base forms have different colours - An example is bromothymol blue with a pK of 7.1 - Universal Indicator is a mixture of coloured indicators with different pKs Lecture 2 Free Energy, Redox Potentials, pH Electrodes

So, if the reaction starts with equal concentrations of 1M of all species A, B, C and D

The Gibbs standard free energy (ΔGo ) is the amount of energy that can be harnessed to do useful work by a reaction that starts under standard conditions (i.e. 1 M concentrations of substrates and reactants) and runs to equilibrium. This equates to the total energy released (the enthalpy, ΔHo ) minus the portion (T.ΔSo ) that is transferred as heat to increase the entropy. Said in another way: ΔGo is the part of the energy released that is available for driving other processes, having adjusted for the entropy changes of the surroundings. A spontaneous reaction has a negative ΔGo ; the more negative the ΔGo , the further the equilibrium favours the products and the more energy is available from the reaction

Food energy is expressed in food calories or kilojoules (kJ). Food calories are 1,000 times greater than the small (gram) calorie. Food calories are therefore kilocalories (kcal). In Europe, these are usually listed in food as kcal but in the USA (and often even in Europe) they are listed just as calories. One food calorie = 1,000 small calories = 1 kcal = 4.184 kilojoules. Calculation of ΔGo from Ke when Protons are Involved: ΔGo ' - A Practical Biochemical Example: The Lactate Dehydrogenase Reaction

Oxidation-Reduction (Redox) Reactions: - When a species loses electrons it is said to undergo OXIDATION. - When a species gains electrons it is said to undergo REDUCTION. - A reaction where the oxidation of one species is coupled to the reduction of another species is called a REDOX reaction For example, consider the simple case of a metal ion in solution that has two oxidation states: M3+ + e- ↔ M2+ -

Imagine that it can exchange electrons with an otherwise inert metal electrode An electrode potential Eh is generated, whose magnitude is dependent on the redox potential of the species involved In order to quantitate the redox potential, we measure it relative to another reaction in a ‘standard reference cell’ that has a fixed, defined potential. The classical standard reference cell is the hydrogen electrode

Reference Electrode Potentials:

Any redox transformation can be represented as a half cell reaction When the two forms of the redox chemical are equal (i.e. where [Xred] = [Xox]) the potential is its midpoint potential, usually represented as Eo or Em. Since many redox reactions are pH-dependent, such values are often quoted for pH 7.0, represented as E'o or Em7.

Redox Potentials and Calculation of ΔGo: - Most redox reactions don’t use hydrogen, but all involve two different redox couples, one of which acts as reductant and the other as oxidant - Each redox couple (Xred/Xox and Yred/Yox) will have its own Em value (measured relative to the Standard Hydrogen Electrode). - The Gibbs Standard Free Energy, ΔGo can be calculated if we know the Em values of the two redox couples

Redox Potentials and Electrochemistry We have discussed measurement of redox potential under standard conditions where the oxidised and reduced species are at equal concentrations (1M). The actual potential generated, Eh , depends on the relative concentrations of the oxidised and reduced species

Hence, if you know the Em of a redox couple and can measure the Eh, you can directly calculate the ratio of oxidised/reduced species. Measuring concentrations using electrodes: - If two reference electrodes are connected together, the potential difference between them is fixed by their individual potential differences. - However, if there is a barrier between them that is permeable only to one specific type of ion, the ion will cause a potential difference across the barrier that is related to the ion gradient. This in turn will create an offset in the potential measured between the two electrodes. This can be calibrated to provide a direct readout of the concentration of the ion. - Electrodes specific for many different types of ion can be constructed in this manner. - The commonest example in a biochemical laboratory is the pH-sensitive glass electrode.

Workshop 1 pH, pK, buffers and equilibria

Lecture 3 Spectrometry 1 SPECTROSCOPY: The study of the interaction of matter with light (in the broad sense i.e. electromagnetic radiation) SPECTROMETRY: The method used to acquire a spectrum SPECTROPHOTOMETRY: Spectrometry in the UV-visible-near infrared range

Photons in the UV/visible/near-IR range cause electrons to move from one orbital to another of higher energy: this is called an electronic transition. This occurs only when the energy of the absorbed photons is equal to the energy required to move the electrons between the orbitals. ~ 700 nm Limits of detection by ~ 400 nm human eye This results in bands of absorption at specific wavelengths by a substance placed in the light path. The wavelengths, shapes and intensities of the bands of absorption are characteristic of the absorbing substance.

Some Biological Examples: - Haem groups have strong bands in the 400-450 nm (Soret or ɣ-bands) and the 550-610 nm (αbands) ranges with wavelengths dependent on haem type and on the chemical state e.g. oxidised, reduced, ligand-bound:

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Tyrosine and tryptophan absorb in the UV at 280 nm. Hence proteins with a known number of Tyr and Trp can be quantitated from their absorbance at 280 nm Nucleic acid bases (DNA) absorb at 260 nm

Absorbance spectroscopy By scanning through the wavelength range of interest and quantitatively measuring the amount of light absorbed at each wavelength it is possible to use spectroscopy for quantitative analyses

Quantitative analysis of absorption requires a spectrophotometer

Selecting the wavelength: - Light dispersion element: prism or diffraction grating - Prism o Typically a triangular glass block o Different wavelengths of light will travel at different speeds, and so the light will disperse into the colours of the visible spectrum, with longer wavelengths (red, yellow) being refracted less than shorter wavelengths (violet, blue)

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o Highly efficient over visible range - transmittance ≈100% Diffraction grating o Surface bearing closely spaced grooves etched on it. o The periodic structure splits and diffracts light into several beams. The groove period must be on the order of the wavelength of interest; the spectral range covered by a grating is dependent on groove spacing o Wavelength dispersion is essentially constant

The sample holder: - Cuvettes or cells The detector: - measuring light intensity - Usually a photomultiplier or photodiode. - They convert a stream of photons into a stream of electrons i.e. light intensity into an electrical current. Quantitative measurements: the beer lambert law: ε, molar extinction coefficient or molar absorptivity (L.mol-1 .cm-1 or M-1 .cm-1 ) C, concentration of absorbing species (in molarity, M) l, pathlength (usually 1cm) How do we use the extinction coefficient, ε, of the Beer-Lambert law to calculate concentrations or rates of reaction? For example, NADH has an extinction coefficient at 340 nm of 6,200 M-1 .cm-1 1. Place the solution in a 1cm cuvette 2. Measure the absorbance at the required wavelength (modern spectrometers can give their output in absorbance units) 3. Calculate concentration using A = εcl i.e. c = A/(ε x l) e.g. If A340 = 0.1, then c (in M) = 0.1/(6200 x 1) Concentration = 1.61 x 10-5 M = 16.1 μM Standard curves of Absorbance versus Concentration: In practice, plots of absorbance versus concentration can deviate from a straight line because of factors such as stray light (this is light that is not at the required wavelength, caused by minor imperfections in the dispersion system)

As Absorbance increases (i.e. as the concentration of species to be measured increases) ‘I’ becomes smaller and the deviation caused by ‘I + s’ becomes progressively greater. The deviation decreases log[(I0 + s)/(I + s)] and so Ameasured becomes progressively less than Atrue. In order to overcome this possible error, we usually use ‘standard curves’ to measure unknown concentrations.

Measuring unknowns with a ‘standard curve’: So, in practice, if we require a measurement over a wide range of concentrations, we usually plot a ‘standard curve’ of Absorbance values using the required range of known concentrations. We can then measure the Absorbance of the unknown sample and read its concentration from the standard curve

Analysing mixtures of two components: Isosbestic points - An isosbestic point is a specific wavelength at which two chemical species have the same extinction coefficient (molar absorptivity), ε. - If the two substances can be interconverted, the absorbance at the isosbestic point will remain constant regardless of the ratio of the two components. - Hence, the absorbance at the isosbestic point can be used to quantitate the total amount of the two species combined, but cannot be used to determine the individual concentrations. If the Beer-Lambert law is obeyed, absorbances are additive. To analyse a two component mixture you need to know the extinction coefficients of each component at two different wavelengths. To be able to calculate the concentrations of each component, they must have a different extinction coefficient at one or both of these wavelengths. You can derive a pair of simultaneous equations with two unknowns (the concentrations of each component) which can be solved to give the concentrations

Rates of reactions with spectrophotometry: If substrates and products have different spectra, the reaction can easily be followed spectrophotometrically Example: NADH but not NAD+ absorbs light at 340 nm and so the rate of interconversion of NADH/NAD+ can be monitored by plotting absorbance at 340nm versus time.

Lecture 4 Spectrometry 2 Fluorescence spectroscopy: When light (photons) is absorbed by some molecules part of the energy is reemitted as light (photons) with a lower energy. The light emitted during fluorescence is at a lower energy than that absorbed and therefore has a LONGER WAVELENGTH Fluorescence methods can be extremely sensitive because of the very low background signal and the ability to detect small numbers of emitted photons Intrinsic and extrinsic fluorescence: - Some molecules have intrinsic fluorescence, e.g. most proteins contain the amino acid tryptophan which can be fluorescent. - These proteins therefore have intrinsic fluorescence - Other molecules may be made fluorescent by adding another molecule that has fluorescent properties – a fluorophore Fluorescent compounds that bind to specific types of molecules can be used to detect them - Fluorescent stains are very sensitive – this is an example of the use of ethidium bromide (intercalator) to detect DNA on a gel - Fluorescent compounds can be covalently linked to proteins - Fluorescent compounds can be attached to antibodies that bind to specific proteins The green fluorescent protein (GFP)

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originally isolated from the jellyfish Aequorea victoria GFP can be attached to proteins of interest by genetic engineering. The GFP gene is spliced onto the gene of a protein of interest. When the protein of interest is expressed, GFP is also covalently attached to it. The GFP module spontaneously folds into its active, fluorescent form, hence allowing the protein of interest to be visualised and located from the GFP fluorescence. GFP itself can also be mutated to fluoresce at different wavelengths – e.g. become red fluorescent protein The folding of this small protein generates a powerful intrinsic fluorochrome or fluorophore. The green fluorescent protein (GFP) Fluorophore combination of three amino acids Ser65-Tyr66-Gly67, found on the α-helix Anti-parallel β-can structure of GFP Tightly packed nature of the barrel excludes solvent molecules, protecting the chromophore fluorescence from quenching by water

Formation of the chromophore during maturation of GFP - Cyclisation - Dehydration - Oxidation

Spectrofluorimetry and quantitative measurements Fluorescence: absorption and emission - Fluorescence emission is normally measured at right angles to the exciting light beam (in a fluorimeter) - Fluorescence light is emitted in all directions so measuring at 90o avoid detection of transmitted and reflected incident light The stokes shift: - The Stokes shift is the difference between positions of the band maxima of the absorption and emission spectra -

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When a system (be it a molecule or atom) absorbs a photon, it gains energy and enters an excited state. One way for the system to relax is to emit a photon, thus losing its energy (another method would be the loss of energy as heat). When the emitted photon has less energy than the absorbed photon, this energy difference is the Stokes shift

The intensity of the fluorescence light emitted is directly proportional to the concentration of the fluorescent molecule (though there are some factors that can alter this linear relation so in practice calibration curves are a good idea); The extent of fluorescence (quantum yield of fluorescence) depends on the nature of the environment of the fluorescing species and the proximity to other molecules. Changes can therefore be used to monitor, for example, the extent of binding of a molecule or a structural change in the vicinity of the fluorophore.

Lecture 5 Spectroscopy 1 Different ways in which light is generated via the excitation of an electron to an excited state and its subsequent return to the ground state 1. Chemiluminescence: Chemical reaction creates the excited state electrons 2. Bioluminescence: Chemiluminescence generated by a biological system 3. Scintillation: Excited state electron generated by the interaction of molecules with a radioactive particle or photon. Used to measure radioactivity in a scintillation counter. 4. Fluorescence and Phosphorescence: Excited state electron generated by absorbance of a photon

What happens to a molecule after it absorbs a photon?

The first, fastest step is (vr). This is the process returns to the ground energy given up is very fast (10-12 s)

vibrational relaxation by which the molecule vibrational state and the turned into heat. It is

The next step is often an internal conversion (IC) process. Internal conversion is the change of an electron from one electronic state to another of the same spin type ( an S to an S) and the energy lost is also given up by heat. It is also very fast (10-12 s)

The electron remains in the S1, v=o state for some time (often 10-9 to 10-8 s). Then one of three events occurs. 1) fluorescence ,F the molecule returns to the ground state and the E lost is given up as LIGHT. 2) Another possibility is another internal conversion, IC. 3) The third possibility is intersystem crossing (IS). In intersystem crossing, the electron changes spin state with or without changing the orbital it is in.

The T1 state is very long lived, on the order of msec to seconds The molecules that entered the T1 state return to the So state via phosphorescence (P), which emits light, or intersystem crossing, IC which results in release of heat.

To summarize, the excited state electron returns to the ground state via a variety of pathways, some of which give off light, but most of which release heat.

What is the difference between Fluorescence & Phosphorescence? - Fluorescence is the name of the light emitted when the S1 state returns to the So state. - Phosphorescence is the name of the light emitted when the T1 state returns to the So state. - Since the S1 state typically only exists for 10-8 s, fluorescence molecules stop emitting light 10-8 s after the light giving rise to absorbance is turned off: - Since the T1 state can exists for seconds, phosphorescent molecules keep emitting light after the light giving rise to absorbance is turned off: Fluorescence Instruments

Emission spectrum: Graph of the intensity of the emitted light as a function of emission wavelength, measured under conditions in which the light exciting the molecule is not varied. Measure by varying the emission monochrometer Tells you what colors of light are being emitted by the molecule. Usually the most Useful for biological and biophysical applications Excitation spectrum: Graph of the intensity of the emitted light as a function of excitation wavelength, measured under conditions in which the wavelength of the emitted light is not varied. Measure by varying the excitation monochrometer Tell you what colors of light are able to cause the molecule to go into the excited state. The excitation spectrum should have the same dependence on l as the absorbance spectrum Technical Details: Corrected vs. Uncorrected Spectra - Unlike absorbance, fluorescence intensity is dependent upon the response of instrumental components as a function of wavelength. - Different instruments give different spectra! - Source...