Spectroscopy is used as a collective term for all the analytical techniques based on the interaction of light and matter. Spectrophotometry is one of the branches of spectroscopy where we measure the absorption of light by molecules that are in a gas or vapour state or dissolved molecules/ions (Tombs, et.al, 1959). Spectroscopy is the use of electromagnetic radiation by a sample in order to identify components of the sample (qualitative analysis) or to measure the amount of material in that sample (quantitative analysis) (Tombs, et.al, 1959). Absorption is a process in which matter captures electromagnetic radiation and converts the energy of the photon to internal energy (Pavia et.al, 2009). Molecules strongly absorb light only in some regions of the electromagnetic spectrum (Tombs et. al, 1959). The spectrum ranges from short wavelengths (x ray and gamma rays) to long wavelengths (microwaves and broadcast radio waves). The electromagnetic wavelength range is between 400nm – 800nm (Pavia et.al, 2009). A photon carries a specific amount of energy defined by it wavelength. Molecules only absorb a photon if the energy it carries matches a certain amount the molecule can use (Clark et.al, 1993). In the Ultraviolet – Visible light (UV-VIS) region of the spectrum this energy corresponds to electronic excitations, which are the promotion of an electron from an occupied orbital to an unoccupied orbital (Pavia et.al 2009).
In this investigation, the aim is to determine unknown protein concentrations from the Bovine Serum Albumin (BSA) standard curve and observe how the protein concentration of BSA and Lysozyme is affected by the addition of detergents, Sodium Dodecyl Sulphate (SDS) and Triton X-100. This study involves two procedures. Firstly a protein assay needs to be carried out in order to investigate the presence of the protein in a series of concentrations of stock solution of BSA, which will be referred to as standards. Plus a protein assay of the two proteins BSA and lysozyme along with the detergents. The assay used in this investigation was the Bradford assay. It is a calorimetric protein assay based on the absorbance shift of the dye Coomassie Brilliant Blue G-250 which forms part of the BIORAD dye reagent (Williams and Fleming, 1980). When used under acidic conditions, the red form of the dye is converted into its blue form in order to bind with the protein being assayed, thus forming a complex (Tombs et.al, 1959). The amount of complex present in a solution is a measure for the protein concentration and is then estimated by use of absorbance readings.
The use of a spectrophotometer will then finalize the experiment as it will determine the absorbance of the standards, the unknowns and the proteins treated with detergent at 595nm. A UV-VIS spectrophotometer has the ability to determine absorption as it consists of two instruments, a spectrometer and a photometer (Clark et.al 1993). The spectrometer uses two light sources, a deuterium lamp for ultraviolet light and a tungsten lamp for visible light. How the spectrophotometer works is that after a light beam bounces off a mirror it hits a diffraction grating. This grating can be adjusted for a specific wavelength. Only a single wavelength can successfully pass through a slight. The spectrophotometer also contains a filter in order to remove any unwanted higher orders of diffraction. The light beam then hits a second mirror before it is split by a half mirror. The beams then pass through the cuvettes and the intensities of the beams are then measured to give the absorbance (Clark et.al, 1993)
Detergents are widely used in protein chemistry, principally to solubilise and stabilize proteins and to disaggregate protein complexes (Rachele et.al, 1994). SDS and Triton X-100 are the most commonly used...