Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
A simple method for the determination of Rubisco in leaf extracts using SDS-PAGE.
Rubisco (ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase) is the key enzyme for photosynthesis and the most abundant leaf protein. Rubisco catalyses two competing reactions, CO2 fixation in photosynthesis and the production of 2-phosphoglycolate in photorespiratory pathways. Rubisco has a low rate of catalysis, and therefore a great deal of N is invested in the Rubisco protein. In C3 species, Rubisco accounts for 15-30% of total leaf N content (Evans 1989, Makino et al. 1992). In many studies, the amount of Rubisco protein is determined by the 14C-CABP (carboxyrabinitol-1,5-bisphosphate, an analog of the putative intermediate of the carboxylation reaction of RuBP) binding technique (Pierce et al. 1980). In contrast, this protocol describes a simple method for Rubisco determination using SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis). Proteins separated by SDS-PAGE are detected by staining, and their amounts are usually determined by densitometric scanning. For Rubisco determination however, colorimetric measurement by eluting the dye has been also used because of the predominant protein on PAGE. Racusen (1973) reported a method for quantitative color measurement involving the elution of Amido Black 10B with 1 M NaOH. Coomassie Brilliant Blue-R-250 (CBB-R) is now widely used for protein-staining. Tal et al. (1985) reported that CBB-R bound to protein on PAGE can be eluted with dimethylsulfoxide. Makino et al. (1985) found that CBB-R can be quantitatively extracted with formamide from SDS-PAGE and that it can be measured spectrophotometrically.
- SDS-PAGE system (containing reagents for SDS-PAGE)
- Centrifuge (cold temperature-controlled if possible)
- Stoppered amber test tubes
- Temperature-controlled incubator equipt with a shaker
Chemicals and Reagents
- 50-100 mM Tris/HCl buffer, pH 7.5
- 200 mM Na-iodoacetate
- 1 M DTT
- 25% (v/v) Triton X-100
- 25% (w/v) lithium dodecyl sulfate
Units, terms, definitions
CBB-R – Coomassie Brilliant Blue-R-250
DS – dodecylsulfate
PAGE – polyacrylamide gel electrophoresis
Rubisco – RuBP carboxylase/oxygenase
RuBP – ribulose-1,5-bisphosphate
SDS-PAGE – sodium dodecylsulfate-PAGE
- Leaf tissues are homogenized at 0 to 4∘C in 50-100 mM Tris/HCl buffer (pH 7.5) containing 5-10 mM DTT (or 0.2-0.8% 2-mercaptoethanol), 2 mM iodoacetate and 5% (v/v) glycerol at a leaf:buffer ratio of 1:5-10 (g:ml). For this extraction, a buffer without sodium or potassium ion is recommended for SDS-PAGE analysis because those cations reduce the solubility of DS (dodecylsulfate). Iodoacetate is an effective inhibitor for protease activity in leaf extracts, and is stimulated in the presence of DS.
- Before centrifugation, a Triton X-100 solution (25%, v/v) is added to a portion of the leaf homogenate to make a final concentration of 0.1% (v/v). An addition of Triton X-100 is very effective for the extraction of Rubisco bound to the membrane fraction (Makino and Osmond 1991).
- The homogenate is centrifugated at 5,000-10,000g for 3 min at 4∘C.
- A lithium DS (LDS, not SDS) solution (25% w/v) and 2-mercaptoethanol are added to the supernatant fluid to a final concentration of 1.0% (w/v) and 1% (v/v), respectively. This preparation is immediately treated at 100∘C for 1 min, and then can be stored at -30∘C until analyzed by SDS-PAGE.
- The sample containing 2-10 μg Rubisco (corresponding to 0.3-1.5 μg chlorophyll) per lane is loaded onto SDS-PAGE containing a 12.5% (w/v) polyacrylamide gel. After electrophoresis, the gels are stained in 0.25% (w/v) CBB-R in 45% (v/v) methanol and 10% (v/v) acetic acid.
- The stained bands corresponding to the large and small subunits of Rubisco are cut out of the gels with a razor blade and eluted in 1.0-2.5 ml formamide in a stoppered amber test tube at 50∘C for 5 h with shaking. The absorbance of the resultant solution is read at 595 nm with a spectrophotometer.
- Rubisco content is determined using the standard curve calculated from the absorbance of known amount of the purified Rubisco.
Notes and troubleshooting tips
When the extraction results of protein-associated CBB-R with formamide were examined, results were independent of the amount of protein put onto the gels, but were affected by the thickness of the gels. When 1 mm thick gels were used it took the dye at least 2 h to completely equilibrate in formamide, and 4 h for 2 mm thick gels. These formamide-CBB-R solutions were quite stable, and their absolute absorbance remained unchanged over 24 h at 50∘C in an amber test tube.
A standard curve should be made with the purified Rubisco because of the large difference in the response of proteins with CBB-R. For example, Rubisco protein shows 10-20% higher absorbance than commercially available BSA (bovine serum albumin) at the same protein content. Thus, protein used as a standard needs to be the same protein that is measured, but it can be calibrated using a correction value between the proteins which has been determined beforehand. The standard curve shows a linear correlation between absorbance of the eluate and the amount of protein put onto the gels, but the stain used repeatedly displays a somewhat upward curvature. This is due to the effect of SDS diffused from the gels while staining. Bradford (1976) reported that the binding of Coomassie dye to proteins is partially interfered with during SDS. However, this has no influence on protein measurement when calibrated with a standard protein.
Bradford MM (1976) Anal Biochem 72: 248
Evans JR (1989) Oecologia 78: 9
Makino A, Mae T, Ohira K (1985) Plant Physiol 79: 57
Makino A. Osmond B (1991) Photosynthesis Res 29: 79
Makino A, Sakashita H, Hidema J, Mae T, Ojima K, Osmond B (1992) Plant Physiol 100: 1737
Pierce J, Tolbert NE, Baker R (1980) Biochemistry 19: 934
Racusen D (1973) Anal Biochem 52: 96
Tal M, Silberstein A, Nusser E (1985) J Biol Chem 260: 9976