The effect that temperatures at 4°C, 23°C, 32°C, and 48°C , pH 3, 5, 7, and 9, the boiled extract, and hydroxylamine had on the peroxidase enzyme extracted from Brassica rapa.
Emma O’Donoghue
Fundamentals of Biology I Lab
Professor William Olsen
October 8, 2015
Abstract
The purpose of this lab was to determine the effects that a number of factors had on enzyme activity. The enzyme used during this lab was peroxidase, which was extracted from the organism Brassica rapa. The peroxidase was standardized at 0.5ml, 1.0ml, and 2.0ml to determine the amount of extract needed for testing, which was one milliliter. The spectrophotometer was used to measure the absorbance in the guaiacol dye to determine the rate of reaction. The …show more content…
The substrate in this reaction is the H2O2. The purpose of this lab is to determine how temperature, pH, boiling, and hydroxylamine effected the activity of the enzyme, peroxidase. Temperature can change the speed with which the substrate and enzyme bind together. When temperatures are higher they move at faster speeds, which could cause the hydrogen bonds to break. The pH values can change the 3D shape of the proteins and, therefore, influence how well the substrate and enzyme bind together. When high temperatures are reach the hydrogen bonds break and the 3D shape is changed. When boiling the enzyme or extract, such high temperature result in a permanent change in the structure of the protein called denaturation (Dolphin, 2015). Competitive inhibitors can affect how easily the substrate can bind to the enzyme. Hypothesis for effect of temperature: As the temperatures increase the enzyme and substrate will collide at a faster rate causing the hydrogen bonds to break and the shape of the enzyme to change. Hypothesis for effect of pH: The enzyme reaction rate of binding to the substrate will favor a more neutral pH value so that the shape of the enzyme is not altered. Hypothesis for effect of boiling on peroxidase: The 3D structure of the protein will permanently change due to denaturation because the high temperatures will cause the hydrogen bonds to break. Hypothesis for probing the active site: Hydroxylamine is a competitive …show more content…
Seven tests tubes were used to find the effect of peroxidase on the rate of reaction. Whether the rate of reaction changed due to the amount of enzyme added was found by mixing two tubes at a time with different measures of peroxidase and recording the absorbance at 470nm on the spectrophotometer in twenty-second intervals. Tube one contained the substrate but no enzyme to be used as a blank, tubes two, four, and six contained the substrate and the dye, tube three contained dilute concentration of peroxidase, tube five contained medium concentration of peroxidase, and tube seven contained concentrated peroxidase. Tubes 2 and 3 were measured with 0.5 ml of peroxidase, tubes 4 and 5 were measured with 1.0 ml of peroxidase and tubes 6 and 7 were measured with 2.0 ml of peroxidase. The results showed that the more peroxidase that was added gave a higher absorbance level in the reaction. Nine tests tubes were used for the effects of temperature and effects of pH on the enzyme activity. Mixing two tubes at a time at four different temperatures and recording the absorbance given in twenty-second intervals found the effects that temperature and pH has on peroxidase. Tubes 2 and 3 were tested at 4°C, tubes 4 and 5 were tested at 23°C, tubes 6 and 7 were tested at 32°C and tubes 8 and 9
Emma O’Donoghue
Fundamentals of Biology I Lab
Professor William Olsen
October 8, 2015
Abstract
The purpose of this lab was to determine the effects that a number of factors had on enzyme activity. The enzyme used during this lab was peroxidase, which was extracted from the organism Brassica rapa. The peroxidase was standardized at 0.5ml, 1.0ml, and 2.0ml to determine the amount of extract needed for testing, which was one milliliter. The spectrophotometer was used to measure the absorbance in the guaiacol dye to determine the rate of reaction. The …show more content…
The substrate in this reaction is the H2O2. The purpose of this lab is to determine how temperature, pH, boiling, and hydroxylamine effected the activity of the enzyme, peroxidase. Temperature can change the speed with which the substrate and enzyme bind together. When temperatures are higher they move at faster speeds, which could cause the hydrogen bonds to break. The pH values can change the 3D shape of the proteins and, therefore, influence how well the substrate and enzyme bind together. When high temperatures are reach the hydrogen bonds break and the 3D shape is changed. When boiling the enzyme or extract, such high temperature result in a permanent change in the structure of the protein called denaturation (Dolphin, 2015). Competitive inhibitors can affect how easily the substrate can bind to the enzyme. Hypothesis for effect of temperature: As the temperatures increase the enzyme and substrate will collide at a faster rate causing the hydrogen bonds to break and the shape of the enzyme to change. Hypothesis for effect of pH: The enzyme reaction rate of binding to the substrate will favor a more neutral pH value so that the shape of the enzyme is not altered. Hypothesis for effect of boiling on peroxidase: The 3D structure of the protein will permanently change due to denaturation because the high temperatures will cause the hydrogen bonds to break. Hypothesis for probing the active site: Hydroxylamine is a competitive …show more content…
Seven tests tubes were used to find the effect of peroxidase on the rate of reaction. Whether the rate of reaction changed due to the amount of enzyme added was found by mixing two tubes at a time with different measures of peroxidase and recording the absorbance at 470nm on the spectrophotometer in twenty-second intervals. Tube one contained the substrate but no enzyme to be used as a blank, tubes two, four, and six contained the substrate and the dye, tube three contained dilute concentration of peroxidase, tube five contained medium concentration of peroxidase, and tube seven contained concentrated peroxidase. Tubes 2 and 3 were measured with 0.5 ml of peroxidase, tubes 4 and 5 were measured with 1.0 ml of peroxidase and tubes 6 and 7 were measured with 2.0 ml of peroxidase. The results showed that the more peroxidase that was added gave a higher absorbance level in the reaction. Nine tests tubes were used for the effects of temperature and effects of pH on the enzyme activity. Mixing two tubes at a time at four different temperatures and recording the absorbance given in twenty-second intervals found the effects that temperature and pH has on peroxidase. Tubes 2 and 3 were tested at 4°C, tubes 4 and 5 were tested at 23°C, tubes 6 and 7 were tested at 32°C and tubes 8 and 9