Determination of chloroplast periplasmic membrane integrity can be used for (1) studies of the molecular mechanisms that promote photosynthesis and (2) detection of cellular activity.

Operation method

Determination of chloroplast periplasmic integrity

Principle

Intact chloroplasts could not carry out the Hill reaction of potassium ferricyanide photoreduction in isotonic medium because potassium ferricyanide could not pass through the periplasm. In contrast, chloroplasts that have lost their intact periplasm, potassium ferricyanide can enter the cytosol for the Hill reaction. According to this principle, by comparing the Hill reaction rates of swollen and unswollen chloroplasts, the degree of integrity of the chloroplast periplasm can be calculated.

Materials and Instruments

Plant material
Potassium ferricyanide NH4Cl
Oxygen Electrode Oxygen Measurement Kit Beaker Microsampler Earwash Balls

Move

1. Take 0.1 ml of chloroplast extract and add it to 0.9 ml of distilled water and stir for 1 min, so that the chloroplasts are swollen by the membrane, and add 1 ml of assay medium Ⅰ for suspension.

2. take 0.1ml of unswollen chloroplast extract and add it into the reaction cup, depending on the volume of the reaction cup, add the measurement medium Ⅱ, add the appropriate amount of potassium ferricyanide and NH4Cl, so that the final concentration of potassium ferricyanide and NH4Cl are 1mmol/L. Stirring, equilibrate for 1min, illuminated to determine the rate of Hill's reaction oxygen release, recorded for 5min, and then clean the reaction cup.

3. Add the swollen chloroplasts into the reaction cup, and then add the appropriate amount of measurement medium Ⅱ, potassium ferricyanide and NH4Cl, so that the final concentration of the reaction system is the same as that of the unswollen chloroplasts measurement system, and the determination process is the same as 2.

4. Calculate the oxygen release per unit time (or Hill reaction rate) in the swollen and unswollen chloroplast assay systems, respectively.

5. Calculate the periplasmic integrity.

Caveat

1. The glass used in this experiment should be stored in the refrigerator, the storage time should not be too long, and it is best to use it now.

2. The separation of chloroplasts should be carried out at 0~4℃, the glassware and solution should be pre-cooled, and the operation should be fast, and the whole separation process should be completed within 5min.

3. The rotational speed of the centrifuge should not be too high during centrifugation, otherwise the starch particles in the chloroplasts will break the chloroplast periplasm.

4. The chlorophyll content of broken chloroplasts and intact chloroplasts in the reaction cup should be equal during the determination of membrane integrity.

Common Problems

The outer and inner membranes of chloroplasts are collectively called the periplasm. The outer and inner membranes of chloroplasts are continuous unit membranes, each with a thickness of 6-8 nm, and there is a 10-20 nm wide gap between the inner and outer membranes, called the membrane gap. Unlike mitochondria, the inner membrane of chloroplasts is not folded inward into cristae, but in some plants, the inner membrane can be folded to form interconnected vesicular or tubular structures, called peripheral reticulum (peripheral reticulum), the formation of this structure can increase the surface area of the inner membrane.

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