A summary of a new technique for the extraction and quantitation of phycoerythrin (PHE) from algal samples is described. Results of analysis of four extracts representing three PHE types from algae including cryptomonad and cyanophyte types are presented. The method of extraction and an equation for quantitation are given. A graph showing the relationship of concentration and fluorescence units that may be used with samples fluorescing around 575-580 nm (probably dominated by cryptophytes in estuarine waters) and 560 nm (dominated by cyanophytes characteristics of the open ocean) is provided

Stewart, D. E.

English

NASA Technical Reports Server

NASA Contractor Report 165996 
A METHOD FOR THE EXTRACTION AND 
QUANTITATION OF PHYCOERYTHRIN 
FROM ALGAE 
Dianne E. Stewart 
THE BIONETICS CORPORATION 
18 Research Drive 
Hampton, Virginia 23666 
Contract NASl-16978 
October 1982 
NI\5I\ 
National Aeronautics and 
Space Administration 
Langley Research Center 
Hampton,Vlrglnla 23665 
NASA-CR-165996 
19830005501 
1111111111111 1111 111111111111111 1111111111111 
NF01909 
OCT 2 61982 
LANGLEY RESEARCH CENTER 
LIBRARY, NASA 
HAMPTON, VIRGINIA 
https://ntrs.nasa.gov/search.jsp?R=19830005501 2020-03-21T05:19:13+00:00Z
TABLE OF CONTENTS 
SECTION PAGE 
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . 1 
INTRODUCTION 2 
METHODS 2 
Extraction of Phycoerythrin . . 2 
Calibration and Analysis 3 
RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 5 
Relationship of Fluorescence Emission to Pigment 
Concentration • • . • . .• . • • • • 5 
Taxonomic Significance of the Phycoerythrin Types . 6 
Selection of Correct Curve for Calculation of 
Plgment Concentration • . . . . 6 
CONCLUSIONS . 7 
REFERENCES 8 
i 
SUMr>1ARY 
This report describes an effective technique for the 
extraction of phycoerythrin from algal cells and prescribes 
a method for quantitating the amount of phycoerythrin pig-
ment. A graph is shown relating concentration to fluorescence 
units. 
INTRODUCTION 
Until now no generally effective method for extract10n of 
phycoerythrin from algal cells has been developed. One problem 
has been the difficulty of d1srupt1ng the cells. Phycobilin-
containing cells tend to be resistant to convent1onal cell des-
truction techn1ques such as grinding. To ensure quantitative 
removal of phycoerythrin (PHE) from algal cells a technique 1S 
needed that combines physical stress with a chemical attack on 
the cell. 
A second problem lies in d1st1ngu1shing and quantitating 
the type of PHE in the sample. Six types of PHE are currently 
known to exist. These forms exh1bit a variety of absorpt1on 
and fluorescence emission character1stics, and d1fferent ex-
tinct10n coeff1cients. It is thus necessary to have some 1dea 
of which p1gment predominates in a sample in order to use 
fluorescence or absorbance to estimate 1tS abundance. 
Th1S report describes an effective technique for the ex-
traction of PHE from algal cells. It also presents a guide for 
distinguish1ng which PHE type predom1nates in a sample and for 
quantitating the extracted PHE. 
METHODS 
Extraction of Phycoerythrin: A precise volume of the culture 
-2-
or sample was passed through a Reeve-Angel No. 984H glass fiber 
filter. Duplicates were run for each sample. Each filter was 
thoroughly homogenized in an all-glass tissue grinder in a 
medium consisting of 0.25 M Trizma Base, 10mM EDTA and 2 mg/mL 
lysozyme (pH adjusted to 5.0 with HCl). The homogenate was 
incubated at 370 C. for 2 hours and then for 10 hours in the 
refrigerator. At the conclusion of incubation the pH was ad-
justed to 7.0 with 0.1 N NaOH, and the total liquid was in-
creased to twice the original volume w~th d~stilled water. 
Filtration on a 5 ~m Millipore filter produced a clear liquid 
for analysis. 
Calibration and Analysis: PHE extracts prepared from several 
cultures were used to determine the relationship between 
fluorescence and absorbance for various PHE types. 
The concentration of a p~gment was determ~ned from its 
absorbance using the following equation: 
absorbance x 1000 ~g/mg 
1% El x pathlength cm 
= ~g PHE/mL extract 
Absorbance was determ~ned at the wavelength of maximum absorp-
tion of an initial PHE solution, using a Cary 17 spectrophoto-
meter. The ch01ce of ext1nction coefficient was based on the 
type of PHE present in the extract, as determined by its ab-
sorpt~on character~stics. The ext~nction coeff1c~ents and 
wavelengths of max~mum absorption of the algal extracts used 
-3-
in thlS study are listed ln Table 1. 
Fluorescence measurements were made on a number of volu-
metric dilutions of each initial PHE solution using a Hitachi 
Perkin Elmer fluorescence spectrophotometer. It was operated 
in the IIratio ll mode to eliminate variations in output due to 
fluctuatl0n of the xenon light source. The excitation wave-
, 
length was set at 520 nm (slitwidth, 20 ~m) and the material 
was scanned from 540 - 600 nm for PHE emission. Emission scans 
were made in an identlcal manner for extracts of seawater 
samples. The temperatures of all PHE Solutl0ns were between 
o 9 and 12 C. during analyses; they were kept in the refrig-
erator at 50 C. at all other times. 
-4-
RESULTS AND DISCUSSION 
Relationship of Fluorescence Emission to Pigment Concentration: 
A graph was prepared from the dilut~on series to relate emis-
sion and concentrat~on for the several types of PHE examined 
(Figure I). Each PHE type produced a line with a different 
slope and a slightly different origin. However, in the fluores-
cence range encountered ~n the seawater samples processed thus 
far (0.2 - 0.7 units) there is relat~vely little variation in 
the corresponding pigment concentration (0.00025 ~g/mL, maxi-
mum) among the three extracts with high fluorescence emission 
wavelength (BL-5, VA-70 and BL-4). BL-l, which fluoresces at 
a d~st~nctly lower wavelength, has a greater slope than the 
three previously mentioned extracts. 
It is not surprising to find variety in the fluorescence/ 
concentrat~on relationship. The extracts do not contain highly 
purified chromophores: rather, the pigments exist in much the 
same protein "environment" as they did within the cell. That 
environment varies from species to species, even for the same 
pigment type. With different pigment types, the variation 
increases. 
The graph produced in this study is a plot of Span 10 
fluorescence units versus PHE concentrat~on (F~gure I). It 
is important to note that these fluorescence units are unique 
-5-
to the fluorescence spectrophotometer, recorder and conditions 
used during the study, and cannot be directly applied to any 
other setup. 
Taxonomic Sign1f1cance of the Phycoerythrin Types: The four 
extracts prepared in th1s study represent three different PHE 
types (Table l). BL-4 and BL-5 are both cryptomonads and con-
tain cryptomonad-PHE 545. The 545 refers to the wavelength of 
maximum absorpt1on by this pigment, 545 nm. Supposedly, there 
are two other types of cryptomonad PHE, 555 and 565 (Siegelman 
and Kyc1a, 1978), but there are no organisms 1n the collectl0n 
wh1ch conta1n these p1gments. Cryptomonad-PHE 545 fluoresces 
at 580 nm. 
BL-l, a cyanophyte, contains a var1ant of b-PHE. This 
p1gment has two absorption peaks (500 and 545 nm) and it 
fluoresces maximally at 560 nm. Accord1ng to Moreth and 
Yentsch (1970), th1s var1ant of R-PHE 1S typical of open ocean 
cyanophytes. 
VA-70, a rhodophyte, contains 6-PHE, which absorbs maxi-
mally at 545 nm and fluoresces at 578 nm. b-PHE 1S confined 
to rhodophytes. However, rhodophytes generally represent a 
very minor component of the phytoplankton. They are more 
common in estuarlne waters than ln the open ocean. 
Selection of Correct Curve for Calculation of Pigment Concen-
tration: The choice of curve may be made on the bas1s of 
-6-
emission wavelength. If the sample fluoresces maximally around 
560 nm, the BL-l curve should be used. This case would be most 
likely if the sample were collected from oceanic waters. 
If, however, the sample fluoresces around 575-580 nm the 
main PHE contribution is probably from cryptophytes, especially 
in estuarine waters. The use of the BL-5 curve is recommended 
in this case. It comes close to passing through the origin of 
the graph, and is similar to the VA-70 curve. 
The wavelength of maximum fluorescence emission for an 
extracted sample can be confirmed by the laser-excited emission 
maximum of the sample. Laser excitation provides a very de-
flned peak. Thus, wavelength of PHE emission will be a valuable 
aid in characterizing phytoplankton populations by remote 
fluorosensors. 
CONCLUSIONS 
The BL-5 curve should be used to determine the pigment 
concentration of samples fluoresclng around 575-580 nm. Such 
samples are probably dominated by cryptophytes in estuarine 
waters. The BL-l curve should be used to determine the PHE 
content of samples fluorescing around 560 nm. These samples 
are dominated by cyanophytes characteristic of the open ocean. 
-7-
REFERENCES 
1. Gantt, E. and C. A. Lipschultz: Phycobilisomes of Porphy-
ridium cruentum I. Isolation. J. Cell. BioI. 54: 313-324, 
1972. 
2. MacColl, R., D. S. Berns and o. Gibbons: Characterization 
of Cryptomonad Phycoerythrin and Phycocyanin. Arch. 
Biochem. Biophys. 177: 265-275, 1976. 
3. Moreth, C. M. and C. S. Yentsch: A Sens~tive Method for the 
Determ~nat~on of Open Ocean Phytoplankton Phycoerythr~n 
Pigments by Fluorescence. Limmo1. Oceanogs. 15: 313-317, 
1970. 
4. S~egelman, H. W. and J. H. Kyc~a: 
Handbook of Phycological Methods. 
chemical Methods, J. A. Hellebust 
(eds). Cambridge U. Press, 1978. 
-8-
Algal B~l~proteins. In: 
Physiological and Bio-
and J. S. Craigie 
I 
I.D 
I 
PHE MAXIMUM MAXIMUM EXTINCTION 
ORGANISM SYMBOL TYPE ABSORBANCE FLUORESCENCE COEFFICIENT (1 em, 1%) 
Unidentified cryptomonad BL-4 Crypt. -PHE 545 nm 580 126 MaeColl et al. 1976 
--
Unidentified cryptomonad BL-5 Crypt. -PHE 545 nm 580 126 MaeColl et al. 1976 
--
Syneehoeoccus sp. BL-1 R-PHE 500, 545 nm 560 74 Moreth and Yentsch, 1970 
Porphyridium purpureum VA-70 b-PHE 545 nm 
, 
578 87.8 Gantt and Lipschultz, 1972 
TABLE 1: CHARACTERISTICS OF THE PHYCOERYTHRIN EXTRACTS USED TO CALIBRATE THE 
FLUORESCENCE SPECTROPHOTOMETER 
3.2 
2.8 
2.4 
2.0 
Cf} 
E-< 
H 
:z; 
::J 
<il 
u 1.6 :z; 
<il 
U 
Cf} 
<il p:; 
0 
::J 
H 
.... 1.2 
0.8 
0.4 
F1gure 1: Graph of the d1lut10n ser1es for the four phycoerythr1n extracts 
used 1n the study. Fluorescence un1ts are span 10 Concentrat1on 
1n flg PHE/l sample by mult1plY1ng by ml of extract/l of sample 
f1ltered. 
.0004 .0008 .0012 .00l6 .0020 .0024 .0028 
10 
1 Report No I 2 Government Accession No 3 Recipient's Catalog No 
NASA CR-165996 I 
4 Title and Subtitle 5 Report Date 
October 1982 A METHOD FOR THE EXTRACTION AND QUANTITATION 
OF PHYCOERYTHRIN FROM ALGAE 6 PerformIng OrganIzatIon Code 
7 Authorls) 8 Performing OrganizatIon Report No 
Dlanne E. Stewart 
t-": __ ----------------------------I 10 Work Unit No 
9 Performing Organization Name and Address 
The Bionetics Corporation 
18 Research Drive 
Hampton, Vlrginia 23666 
11 Contract or Grant No 
NASl-16978 
t::~---~---:-:------------------------l13 Type of Report and Period Covered 
12 Sponsoring Agency Name and Address Contractor Report 
National Aeronautics and Space Administration 
Washington, D.C. 20546 
14 Sponsoring Agency Code 
15 Supplementary Notes 
Langley Technical Monitor: Dr. Reginald J. Exton 
16 Abstract A summary of a new technique for the extraction and quantltation 
N-305 
of phycoerythrin (PRE) from algal samples is descrlbed. Results of 
analysis of four extracts representing three PHE types from algae 
including cryptomonad and cyanophyte types are presented. The 
method of extraction and an equatlon for quantitatlon are given. 
A graph showing the relationshlp of concentration and fluorescence 
unlts that may be used with samples fluorescing around 575-580 nm 
(probably dominated by cryptophytes in estuarine waters) and 560 nm 
(domlnated by cyanophytes characteristic of the open ocean) is 
provided. 
For sale by the National Technical Information SerVice, Springfield, Virginia 22161 
End of Document 


A method for the extraction and quantitation of phycoerythrin from algae

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