PREFACE: Estimation of RPE Mass

Quantities of RPE 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 RPE mass are thus performed on the basis of an 8.2 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., [RPE] = 0.122 x A₅₆₅

PART A. Determination of Concentration and Absorbance Ratios

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.  The diode array spectrophotometer used internally measures absorbance at 2 nm intervals; for this reason, determinations and calculations are done on the basis of A₅₆₆ rather than A₅₆₅ .  Differences between the two values are extremely small and do not materially impact the results reported.

2. The RPE sample is diluted, in triplicate, with potassium phosphate buffer (KPB) to achieve final concentrations of approximately 1,000, 250, and 80 µg/mL.  All dilutions are recorded.  If the RPE sample is in the form of a suspension in ammonium sulfate, mix the sample well before performing the initial dilution.

3. With KPB in 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.

4. The absorbance of the ~1,000 µg/mL dilution is read at 620 nm.  Absorbance readings will be low (<0.1), but it is our experience that less dilution results in a solution that is too turbid to read.  The absorbance reading is multiplied by the dilution factor to obtain A₆₂₀.

5. The absorbance of the ~250 µg/mL dilution is read at 280 nm.  The absorbance is multiplied by the dilution factor to obtain A₂₈₀. Absorbance readings should be in the range of 0.2 to 0.8 to obtain accurate readings; if not, a more concentrated or more dilute sample is prepared as required to obtain an absorbance in the range.

6. The absorbance of the ~80 µg/mL dilution is measured at 566, and 496 nm. The absorbance reading at 566 nm should be in the range of 0.3 to 0.8 for accurate readings; if not, new dilutions, adjusted appropriately, are prepared.  The absorbance readings are multiplied by the dilution factor to obtain A₅₆₆ and A₄₉₆.

7. The readings are repeated with each of the triplicate samples and averages for A₆₂₀, A₅₆₆, A₄₉₆, and A₂₈₀ are obtained.

8. The concentration of RPE in the original sample is calculated as:
 

[RPE] = 0.122 x A₅₆₆

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

A₅₆₆/A₂₈₀ > 5.30

A₅₆₆/A₄₉₆ < 1.5

A₆₂₀/A₅₆₆ < 0.005
 

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, uv quartz glass.

5. Spectrofluorometer cells, 1 cm path length, uv 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. The 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 RPE sample is further diluted to approximately 0.5 µg/mL with KPB in a single dilution in a 25 - 100 mL volumetric flask.  Three separate dilutions are prepared for triplicate measurements.

4. The spectrofluorometer excitation wavelength is set to 565 nm.  Slit width for both excitation and emission monochromators are set to 3 nm.  (This is important: slit width settings will materially effect the appearance of the peaks.) Emission spectra are then scanned from 450 - 750 nm.  A Raman scattering peak at the wavelength of excitation may be present.

5. The process is repeated for excitation wavelengths of 490 and 545 nm.  The emission spectra obtained are printed as appropriate for the equipment in use and added to the certification data packet.
 

PART C. Polyacrylamide Gel Electrophoresis
 

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.
 

NOTES:
 

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 RPE 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

 
(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.