Beta-galactosidase enzyme reaction rates under different condition of temp.

and competitive reactive sites

Coy Guffey

Biology 121, Section 01 Professor Mayers September 14, 2011 Introduction The bacterium E. coli produces an enzyme called beta-galactosidase. E. coli prod uces and uses this enzyme as a catalyst to break the chemical bonds of the disac charide lactose into the monosaccharide forms of galactose and glucose. Accordin g to an article Gene induction: -galactosidase in E. coli, Lactose is a sugar found i n milk. E. coli produces this enzyme that creates a su strate known as glucose u sed in respiration. B-galactosidase is normally switched off except in the prese nce of lactose 2011. Beta-galactosidase reacts to lactose ecause of the specific ally shaped surface area of the disaccharide. This reaction is demonstrated y t he following equation: (Eq.1) Beta-Galactosidase + Lactose = Galactose + Glucose EQ.1Introduction to Enzyme Kinetics: Assay of -galactosidase To artificially cause this reaction, we have used the compound o-nitrophenyl gal actoside (ONPG) instead of lactose. ONPG has a similar receptor site and will ca use the eta-galactoside to react chemically to reak down the ONPG the same way it reacts to lactose. This reaction is demonstrated y the following equation: (Eq.2) Beta-Galactosidase + ONPG = O-nitrophenol + Glucose EQ.2 Introduction to Enzyme Kinetics: Assay of -galactosidase The use of ONPG will facilitate the need to record the a sor ance y use of the spectrophotometer. The end result of the chemical reaction is yellow in color an d therefore allows the spectrophotometer to record the concentration of the rea kdown of chemical onds on its way to equili rium. The first experiment is roke n down into three solutions to o serve the chemical reaction under different tem perature conditions. The first solution is 3ml of 2mM ONPG with the eta-galacto sidase enzyme added every minute for 10 minutes y the spectrophotometer at room temperature, or roughly 22 degrees Celsius. The second solution is 3ml of 2 mM ONPG with the enzyme added to e recorded y the spectrophotometer at 4 degrees Celsius every minute for 10 minutes. The third solution is 3ml of 2mM ONPG with the enzyme added, to e recorded y the spectrophotometer at 37 degrees every mi nute for 10 minutes. The second experiment is to determine the competitive aspects or if at all of la ctose vs ONPG for the receptor site of eta-galactosidase enzyme. The first solu tion, used in this experiment, is a control solution of 1.5ml Buffer and 1.5ml o f 2mM ONPG solution to e recorded y the spectrophotometer every minute for 10 minutes at room temperature. The second solution is 1.5ml of Lactose and 1.5ml o f 2mM ONPG solution to e recorded y the spectrophotometer every minute for 10

minutes at room temperature. I hypothesize that in the first experiment, the second solution tested at 4 degr ees Celsius will meta olize slower than the first solution at room temperature. The lower temperatures will lead to a slower concentration rate. This is ased o n the fact that with a reaction at an am ient temperature that is lower, the rea ction will e lower as a result. The third solution at 37 degrees Celsius will m eta olize faster due to the increased am ient heat leading to a faster equili ri um. In the second experiment, the control solution having only the amount of 1.5ml of ONPG to react with and there for the enzyme the reaction rate should e very different. The lactose and ONPG solution should produce more then control solut ion y half. The lactose and ONPG solution should react much like the first solu tion from the first experiment ecause ONPG and Lactose have similar receptor si tes on their surface area.

Methods First Experiment: Solution 1: We cali rated the spectrophotometer with the uffer solution and set the wavelength to 420mM, we captured our cuvette of 3ml ONPG of 2mM with enzyme . The solution was stirred and tested in the spectrophotometer and then tested o nce every minute for 10 minutes while recording the o tained data. Solution 2: We placed the cuvette in the freezer at 4 degrees Celsius for five m inutes, we o tained the 3ml ONPG of 2 mM with the enzyme in it. We stirred the s olution and wiped down the cuvette to minimize disruption in the testing. (Witho ut recali rating). Then, we repeated this process for 10 minutes replacing the s olution in the freezer etween testing while checking the results on the spectro photometer every minute. Solution 3: We then o tained 3 ml ONPG of 2mM with enzyme, stirred the solution, wiped it down, and tested it on the spectrophotometer (without recali rating). Then, we placed it in the incu ator at 37 degrees Celsius. We tested it every mi nute for 10 minutes while replacing it in the incu ator etween checks. Second Experiment: Solution 1 (Control): We o tained 1.5ml of ONPG of 2 mM and 1.5ml of Buffer with enzyme in it as our control. We stirred it, wiped it down, and then tested it i n the spectrophotometer. Without recali rating, we tested it every minute for 10 minutes. Solution 2: We o tained our solution of 1.5ml ONPG of 2 mM and 1.5ml of Lactose with enzyme in it, stirred it, wiped it down, and tested it in the spectrophotom eter. (Without recali rating) we tested it every minute for 10 minutes. Results First Experiment Solution 1: (room temp.)The increase in percentage was somewhat larger, ranging from 0.07% to 0.34% at an average rate of 0.03% per minute.(fig .1) Solution 2: (4 degrees Celsius) The increase in percentage was a mild change, in creasing from 0.07% to 0.20% at an average rate of 0.01%-0.02% per minute.(fig.1 ) Solution 3: (37 degrees Celsius) There was a large increase in concentration ran ging from 0.07%-0.52% at an average rate of 0.04% per minute.(fig.1) Second Experiment Solution 1 (Control): The solution increased from 0.12% to 0.05%, increasing in concentration y 0.03% for the first six minutes and then increased y 0.05% for the following four minutes. Solution 2: The solution increased from 0.11% to 0.54%, increasing in concentrat ion y an average of 0.03% for the first two minutes, y 0.05% for the next thre e minutes, and then y 0.06% for the last five minutes. Discussion My hypothesis for the first experiment was correct; the lower temperature affect

ed the production of o-nitrophenol, causing a lower percentage in concentration over the 10 minute period. The room temperature solution increased somewhat due to the higher am ient temperature, causing the percentage of o-nitrophenol conce ntration to increase per minute over the 10 minute period, giving it a higher co ncentration rate. The solution tested at 37 degrees Celsius showed an increase i n o-nitrophenol a ove that of room temperature, showing that the excess heat att ri uted to the rise in concentration. In the second experiment, I was not correct in my hypothesis. My data collected from my control of ONPG and Buffer solution showed an increase in concentration of o-nitrophenol production. These results, though different from minute to minu te, concluded almost the same results as my solution of ONPG and Lactose. The da ta shows that the addition of 1.5ml of lactose didnt, in fact, decrease the overa ll production of o-nitrophenol. It is possi le a mistake was made due to human e rror or the fact we did not recali rate the spectrometer. To confirm or deny the data of the experiment should e repeated multiple time (recali rating after ea ch solution data is recorded) to weed out any possi le error in data collection or in the experimental processes. The actual result of the lactose and ONPG solu tion should have een a lesser concentration of o-nitrophenol to e recorded y the spectrometer. This would have accounted for the percentage of enzyme collect ed y the lactose. Gene induction: -galactosidase in E. coli. Practical iology. The Nuffield Foundati on & Society of Biology, 2011. We . Introduction to Enzyme Kinetics: Assay of -galactosidase . Science AAAS. N.p.n.d. W e . Series 1: Solution tested at 4 degrees Celsius (freezer) Series 2: Solution tested at 22 degrees Celsius (room temp.) Series 3: solution tested at 37 degrees Celsius (incu ator) Series 1: Solution with uffer and ONPG (control) Series2: Solution with lactose and ONPG (experiment)