BIo 1AL- lab 5 (photosynthesis and vibrio colony restreak)

chloroplast vs thylakoid membranes

• pigments (chlorophyll a, b, and carotenoids) located in thylakoid membranes

flowchart list of photosynthesis inc. reactants and products

• light rxns in thylakoid membrane:

  • hill rxn: H20 + NADP⁺+ ADP + Pi —> ½ O2 + 2(NADT + H⁺) + ATP

  • PSII, Pq, Cyt, Pc, PSI, Fd, NADP⁺ reductase

describe the series of chemical rxns in chloroplasts that lead from absorption of light to transfer of E- from water to NADP and production of both O2 and ATP

explain each step of the chloroplast isolation procedure: use of blender on spinach leaves, filtration with cheese cloth, pelleting chloroplasts, use of suc-phos, and phos buffers, and generating standardized concentrations of chloroplasts

• blend spinach w sucrose-phos buffer (chloroplasts stay intact due to sucrose osmoticum)

• pour off supernatant to leave pellet of chloroplast

• tube A: add acetone to extract pigments

  • supernatant II includes pigments and used for paper chromatography

• tube E: resuspend pellet with cold suc-phos buffer

  • add E to tube L (phos only buffer) and spec for absorbance

  • calculate and create standardized chloroplast soln (0.1 mg Chl/mL

explain the role of controls when testing drugs

provide a baseline for comparison to ensure that any observed effects are truly caused by drug being tested

understand how paper chromatography works to permit isolation of pigments, and identify chlorophylls a and b, carotenoids, and carotene on your chromatogram

pigments partition betwen hydrophilic paper and hydrophobic developing solvent

• polar pigments migrate a SHORTER distance

• hydrophobic pigments migrate a FARTHER distance

• bottom to top (polar to nonpolar) : Chl b, Chl a, carotenoids, carotene

calculate an Rf value

RF = migration distance of substance/ migration distance of solvent(solvent front)

absorption/action spectrum of a mixture of pigments

graph of the amount of light absorbed by a given substance at various wavelengths

DCPIP vs DCMU vs methylamine

• DCPIP- artificial e- acceptor (end of ETC), used to assess rate of photosynthesis bc it loses blue color as it recieves e-

  • correlates to the rate of e- transfer from H20 to PSII, the ETP, PSI, and the reductase

• DCMU- uncharged hydrophobic, inhibits photosynthesis by blocking e- flow from PSII to Pq

• methylamine- uncharged weak base, enters lumen to bind to H+ in thylakoid

  • adding it should speed rates of ETC (loss of blue color)

how to make a standardized chloroplast soln

• 0.0533 x absorbance “L” x 36 = mgChl/mL in “E”

• CxVx = XyVy to calculate Vx mL to add to make standardized soln

  • Cx- “E”

  • Cy- 0.1 mgChl/mL

  • Vy- 5mL buffer + Vx mL

• add 5 mL cold suc-phos buffer and add Vx mL of tube E

effect of phosphate buffer

lack of sucrose lyses chloroplasts allowing us to add a loss of color e- acceptor (DCPIP)