Synopsis of Experiment
The effect of pH, temperature, or substrate concentration on sucrase activity will be assessed in experimental [active sucrase present] and control [active sucrase absent] sucrose solutions by measuring the product [glucose or fructose] produced. The following process is used.
Sucrose solutions – with and without active sucrase – are hydrolyzed during a 20-minute incubation. Following incubation, DNS (dinitrosalicylic acid) is added. The pH of the DNS solution denatures sucrase and stops the reaction, thus fixing the concentration of the reaction products.
After heating the solutions to 90°C (194°F), DNS will convert to ANS (3-amino-5 nitrosalicylic acid) if a monosaccharide – like glucose or fructose – is present. The amount of ANS, a reddish-brown colored chemical, is determined using a spectrophotometer, an instrument that measures the amount of light that is transmitted through a colored solution. The more ANS in the solution, the greater the optical density will be. The amount of ANS produced is analogous to the amount of product produced from the enzyme reaction.
The effect of pH on sucrase activity in sucrose solutions with varying pH values from 3.0 to 9.0 will be assessed.
The effect of temperature on sucrase activity in sucrose solutions at varying temperatures from 10°C (50°F) to 70°C (158°F) will be assessed.
The effect of substrate concentration on sucrase activity in solutions with different concentrations of sucrose will be assessed.
Predictions
Based on what you know, predict the outcome of the experiment. Complete the numbered statements below by choosing one response for each number.
When you are done, click the Submit button to save your responses to the laboratory report.
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Sucrase will have the greatest activity at
pH 7
Sucrase will have the greatest activity at
10 °C (50 °F)
Sucrase activity
does not change with increasing sucrose concentration
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Variables
Now determine the dependent, independent, and controlled variables for each experiment in this laboratory activity by clicking on the appropriate answers.
Check your answers until you have chosen all variables correctly. There will be one choice for Dependent and Independent Variables and multiple choices for Controlled Variables. When you are done, click the Submit button to save your responses to the laboratory report.
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Effect of pH on Enzyme Activity
Dependent Variable
amount of product (glucose and fructose) produced
Independent Variable
pH
Controlled Variables
temperature
amount of substrate (sucrose) present
sucrase + sucrose incubation time
Effect of Temperature on Enzyme Activity
Dependent Variable
amount of product (glucose and fructose) produced
Independent Variable
temperature
Controlled Variables
pH
amount of substrate (sucrose) present
sucrase + sucrose incubation time
Effect of Substrate Concentration on Enzyme Activity
Dependent Variable
amount of product (glucose and fructose) produced
Independent Variable
amount of substrate (sucrose) present
Controlled Variables
temperature
pH
sucrase + sucrose incubation time
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