PREFACE - Estimation of Allophycocyanin (APC) Mass

PART A - Determination of Concentration and Absorbance Ratios

PART B - Fluorescence and Absorbance Spectra

PART C - Polyacrylamide Gel Electrophoresis

NOTES

PREFACE: Estimation of APC Mass

Quantities of APC present in solutions are conventionally estimated based on the light absorbance of the solutions rather than by direct gravimetric determination. Gravimetric determinations are destructive of the sample used and require relatively large quantities of material to achieve suitable precision, while spectrophotometric methods are nondestructive, sensitive, highly specific, and very precise when performed with appropriate care. Estimation of mass in this manner has become a de facto standard for producers and consumers of phycobiliproteins. Estimates of APC mass are thus performed on the basis of 7.3 cm² mg^-1 absorptivity. While the units of this value can be confusing, the end result is that concentration of a solution can be calculated by dividing absorbance, at an appropriate dilution, by the absorptivity (or multiplying by the inverse of the absorptivity); i.e.,
 

[APC] = 0.137 x A₆₅₂

Equipment and Supplies

1. Spectrophotometer.

A good quality instrument with precision to at least three decimal places is required. (Hewlett-Packard 8452A used internally.)

2. Automatic pipettes, ranges as required; tips.

3. Spectrophotometer cells, 1 cm path length, uv quartz glass.

Materials

1. Potassium phosphate buffer, 50 mM pH 7.0 (see below)

PROCEDURE

1. The spectrophotometer is prepared for operation following manufacturer's instructions. Operator verifies that instrument is or has been calibrated as specified by manufacturer's and internal procedures.

2. With KPB in a quartz spectrophotometer cell, the spectrophotometer is blanked. Blanks should be repeated throughout the procedure as required. A blank should be read prior to each reading unless procedures and experience indicate that less frequent blanking is acceptable.

3. The APC sample is diluted with potassium phosphate buffer (KPB) to achieve a final concentration of approximately 80 µg/ml. If the APC sample is in the form of an ammonium sulfate suspension, mix the sample well before performing the initial dilution.  Dilutions are made in triplicate, but each dilution should be prepared immediately prior to reading; absorbance values of APC will decrease over time at the low concentrations required for spectrophotometric observations. All dilutions are recorded.

4. The absorbance of the ~80 µg/ml dilution is measured at 652, 620 and 280 nm. The absorbance reading at 652 nm should be in the range of 0.3 to 0.7 for accurate readings; if not, new dilutions, adjusted appropriately, are prepared. The absorbance readings are multiplied by the dilution factor to obtain A₆₅₂ and A₆₂₀ and A₂₈₀.

5. The readings are repeated with each of the triplicate samples and averages for A₆₅₂, A₆₂₀, and A₂₈₀ are obtained.

6. The concentration of APC in the original sample is calculated as:
 

[APC] = 0.137 x A652

The following values are calculated and compared to those given in the Finished Product Specification to ensure that they meet the specified criteria:
 

A₆₅₂/A₂₈₀

A₆₅₂/A₆₂₀

EQUIPMENT AND SUPPLIES

1. Spectrophotometer.

A good quality instrument with precision to at least three decimal places is required. (Hewlett-Packard 8452A used internally.)

2. Spectrofluorometer.

(Perkin Elmer LS5B used internally.)

3. Automatic pipettes, ranges as required; tips.

4. Spectrophotometer cells, 1 cm path length, quartz glass.

5. Spectrofluorometer cells, 1 cm, quartz glass.

6. Volumetric flasks, 25 - 100 mL.
 

MATERIALS

1. Potassium phosphate buffer, 50 mM pH 7.0 (see below)
 

PROCEDURE

1. The spectrophotometer is blanked as above (A.3.).

2. A sample diluted to ~80 µg/ml in A2 is loaded into a quartz spectrophotometer cell, placed in the spectrophotometer and scanned from 250 nm to 750 nm, with readings at 2 nm intervals. The absorbance spectrum obtained is printed as appropriate for the equipment in use and added to the certification data packet.

3. The APC sample is further diluted to approximately 1 µg/ml with KPB in a single dilution in a 25 - 100 ml volumetric flask. Three separate dilutions are prepared for triplicate measurements, but again, as above, samples are prepared immediately before reading, since the fluoresecnce of APC will decline over time at low concentrations.

4. The spectrofluorometer excitation wavelength is set to 645 nm. (The excitation wavelength slightly lower than the excitation maximum to allow an emission spectrum free of Raman bands to be observed.) Slit width for both excitation and emission monochromators are set to 3 nm. (NOTE: slit width settings will materially effect the appearance of the peaks.) Emission spectra are then scanned from 650 - 750 nm.

5. The spectrofluorometer emission wavelength is set to 665 nm. Excitation spectra are scanned from 250 - 650 nm.

6. The excitation and emission spectra are plotted as appropriate for the equipment utilized.
 

Electrophoresis apparatus and techniques vary widely between laboratories. ProZyme's tests are run in "tube gels", which we have found to have the highest sensitivity and resolution with phycobiliproteins. The tris-glycine buffer system is used, with a 4% stacking gel, pH 6.8, and a 7.5% running gel, pH 8.8. Quantitation is performed with a scanning densitometer (Hoefer Scientific Instruments Model GS 300). ProZyme will provide detailed procedures for these specific apparatus on request.

NOTE: Gradual dissociation of APC into its component subunits may occur during the course of electrophoresis, leading to "tailing" of the APC band. The degree of tailing is highly sensitive to running conditions; maintaining temperature of the gel at 0 - 5C helps to minimize this process. Under these conditions, tailing should not be so severe as to significantly impair densitometric determinations of relative protein abundance.

1. Waiting time.

When diluted to low concentrations, phycobiliproteins will gradually dissociate into their component subunits, altering the spectral and fluorescence properties of the solution. It is important that measurements be taken shortly after dilution to obtain valid results. This is most critical for samples with the highest dilution factors.

2. Relative Quantum Yield.

In addition to the above factors, ProZyme routinely reports to its customers a value for "Relative Quantum Yield", which is a measure of the fluorescence of the product relative to a standard. This measurement is taken to assure that the fluorescence of the molecule has not been compromised in any way--that its "brightness" is typical of what is expected. It is our experience, however, that the transfer of this measurement technique between laboratories can only be accomplished after substantial cross-calibration and standardization. This is due to inconsistency in standard materials and to the lack of standardized reporting units for fluorescence. We recommend that customers determine and record for each lot received the ratio of APC fluorescence to absorbance in the units reported by their individual fluorometer and consult with ProZyme should any "outlying" values be obtained.

3. Potassium phosphate buffer (KPB)

To make 1 liter:
 

6.80 g Monobasic potassium phosphate (KH₂PO₄)

1.90 g Potassium hydroxide (KOH)

Make up to 1 Liter

With good quality deionized water, the pH will be very close to 7.00. Any adjustments in pH that are required are made with:
 

(i) 50 mM potassium phosphate solution (6.80 g/l without added KOH) to lower the pH, or

(ii) 0.1 M potassium hydroxide, added dropwise, to raise the pH.