Sandbox323: Difference between revisions
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=== Spectrophotometry === | === Spectrophotometry === | ||
[[Image:Ice_bath_basic_conditions_with_salt.jpg| | [[Image:Ice_bath_basic_conditions_with_salt.jpg|500px|left|thumb|4 degrees C reaction under basic conditions]] | ||
50uL of purified protein from the first elution was added to 3mL of cold 1mg/mL PNPA. Solid sodium chloride was added to achieve a final concentration of 5mM NaCl. The absorbance peak was measured every 60 seconds for 30 minutes via a Vernier Go Direct SpectroVis Plus Spectrophotometer. Peak maximum was located at 427.8nm. The sample incubated in an ice bath between measurements. No discernible relationship between time and absorbance was noted. pH of the solution was 10.60. The instrument to measure pH was Vernier Lab Quest 3 with Tris-compatible flat pH sensor calibrated with two points. Theorizing the enzyme would increase activity at the physiological pH of common prokaryotes, we lowered the pH with 2M HCl. 2M NaOH was used to raise the pH due to the weak buffering capacity of the PNPA solution near pH 7. Final pH of the 1mg/mL PNPA solution was 7.30 | 50uL of purified protein from the first elution was added to 3mL of cold 1mg/mL PNPA. Solid sodium chloride was added to achieve a final concentration of 5mM NaCl. The absorbance peak was measured every 60 seconds for 30 minutes via a Vernier Go Direct SpectroVis Plus Spectrophotometer. Peak maximum was located at 427.8nm. The sample incubated in an ice bath between measurements. No discernible relationship between time and absorbance was noted. pH of the solution was 10.60. The instrument to measure pH was Vernier Lab Quest 3 with Tris-compatible flat pH sensor calibrated with two points. Theorizing the enzyme would increase activity at the physiological pH of common prokaryotes, we lowered the pH with 2M HCl. 2M NaOH was used to raise the pH due to the weak buffering capacity of the PNPA solution near pH 7. Final pH of the 1mg/mL PNPA solution was 7.30. | ||
=== Experimental Data and Discussion === | |||
[[Image:Absorbancelongph7cold.png|500px|left|]] | [[Image:Absorbancelongph7cold.png|500px|left|]] | ||
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The cold 4 degrees C reaction was changed to room temperature 20 degrees C. All other conditions remained constant to evaluate the effect of temperature on enzyme. Temperature increase negatively affects enzyme performance. Considering the cold loving nature of Chitinophaga pinensis, the enzyme being more active at a lower temperature is a reasonable conclusion. | The cold 4 degrees C reaction was changed to room temperature 20 degrees C. All other conditions remained constant to evaluate the effect of temperature on enzyme. Temperature increase negatively affects enzyme performance. Considering the cold loving nature of Chitinophaga pinensis, the enzyme being more active at a lower temperature is a reasonable conclusion. | ||
[[Image:Iceenzymeefficiencyscinotation.png|600px|left|thumb|Enzyme Activity at neutral pH at cold temperature with Chloride cofactor]] | |||
[[Image:Roomtempconcentrationtime.png|600px|left|thumb|Enzyme Activity at neutral pH at room temperature with Chloride cofactor]] | [[Image:Roomtempconcentrationtime.png|600px|left|thumb|Enzyme Activity at neutral pH at room temperature with Chloride cofactor]] | ||
