UV-VIS Spectroscopy/Steady State Enzyme Kinetics
Completed October 1,2012
Submitted on 0ctober 19,2012
Bazinet Christine TA.
Spectroscopic analysis is a technique which examines a solutions tendency to absorb light. Two experiments were performed to help demonstrate Beer-lamberts law as well as gain understanding of Michaelis- Menten Kinetics. The first experiment tested four samples, with known concentrations to calculate the absorbance which corresponded to the known volumes. An unknown concentration was determines based on a curve constructed of the absorbance vs. concentration of the other diluted standards. An absorbance of 0.2741AU was determined for the unknown with a molar concentration of 2.69 x10 -5M . The second experiment monitored the absorbance of the p-nitrophenyl acetate being converted to p-nitrophenol over time. The turnover rate of chymotrypsin, k2 is found to be different in all 6 reactions with the highest turnover rate with 2mM solution of P-nitrophenyl acetate with 50μl of chymotrypsin at 5.85x10-3 M and the lowest turnover rate with 2mM solution of p-nitrophenyl with 25μl of enzyme at 5.18x10-4M. This lab demonstrated that Concentration verse absorbance is a linear function and the rate of the reaction is only dependent on the enzyme, and not on the substrate.
A specrum of rays omitted from the sun, sunlight, can be categorized into three categories; Ultraviolet, visible and infrared. These ranges of the spectrum are unique in wavelength and frequency. Wavelength is the distance between two peaks and frequency is that number of cycles which occurs given a certain time frame. Colors perceived by the human eye are wavelengths in the visible spectrum. The visible spectrum ranges from 400nm violet, to 800 nm, red. However the visible spectrum is a very small part of the electromagnetic spectrum. Other parts consist of gamma, x, infrared, microwave and radio rays. Light that passes through or reflect off an object is complementary to the wavelength absorbed. The absorption can be recorded and compared to a physical law known as Beer-Lambert law of absorption. This law provides insight on the absorption process as well as, a finite absorption based on the path length of the sample, concentration and the absorptivity per mole. 1 A=εbc
A;Absorption, ε; Molar Absorptivity, c;concentration, b;path length. In order to calculate the absorption an equation which related the transmittance to the absorbance will be used A=-logT
Beer’s law falls apart when the solutions are very concentrated however. It is most useful for very dilute solutions, scattering interferences with readings at high concentrations. High intensity incident radiation is also determined because it introduces secondary interactions between the molecule and the light which affects the readings.
Michaelis-Menten Kinetics describe the rate of enzyme catalyzed reactions. An enzmeme being catalyzed can be broken into three major interactions. S+E↔SE→E+P
S; substrate, E;enzyme, P; product , SE ;intermediate complex. The substrate and the enzyme form an intermediate complex which is converted back into enzyme and product. The second arrow indicated the rate of the second step which is the rate determining step in an enzyme catalyzed reaction. The rate of the reaction can be expressed as,
t;time, [Eo] ; initial concentration of enzyme, Km;Michaelis constant. We must assume that [S] >>>Km so this constant is eliminated. The equation then can be simplified too 3
With [P] equal to A/e the equation can be rewritten as
Michaelis-Menten Kinetics carry some assumptions. It assumes that the rate of change of [ES] is very small compared to the rate of change of [p] and [S], which results in the variable...
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