The presence of Pb(++), Co(++), Cu(++), and Cd(++), each of which possesses a certain luminescence-triggering activity of aequorin, potentially interferes with the specificity of the aequorin luminescence response to Ca(++). Interference by the above cations can be eliminated, without influencing the sensitivity of the luminescence of aequorin to Ca(++), by adding 1 mM of sodium diethyldithiocarbamate

Johnson, F. H.

Shimomura, O.

English

NASA Technical Reports Server

SP%ECBF!CITY OF AEQUORlN LUMINESCENCE 
'PC) CALCIUM 
Osamu Shimomura and Frank H. Johnson 
Biology Department 
Princeton University 
Princeton, New Jersey 
SUMMARY 
The presence of Pbf+, Co*, Cu++, and Cdf+, each of which possesses a 
certain luminescence-triggering activity of aequorin, potentially interferes 
with the specificity of the aequorin luminescence response to Ca++. Inter- 
ference by the above cations can be eliminated, without influencing the 
sensitivity of the luminescence of aequorin to Caff, by adding 1 mM of 
sodium diethyldithiocarbamate. 
INTRODUCTION 
The isolation of a substance involved as an active principle in the lumines- 
cence of Aequorea aequorea, a hydromedusan jellyfish indigenous to 
coastal waters of the Pacific Northwest, was reported some years ago 
(Shimomura et al., 1962). The purified substance proved to be a conjugated 
protein for which the name aequorin was introduced. Later studies on other 
bioluminescent organisms, Chaetopterus and Meganyctiphanes (Shirnomura 
and Johnson, 1966; 1968), indicated that aequorin was the first example of 
a type of luminescence system that was given the collective name "photo- 
protein," characterized by the properties that (1) under optimum condi- 
tions the total amount of light emitted was proportional to a specific pro- 
tein, and (2) the Wt-emitting process did not directly involve any enzyme, 
at least in the usual sense of the term. 
In aqueous solution, aequorin was found to have the unique property of 
being triggered by traces of Ca++, and to a lesser extent by Srf+, to emit 
light, either with or without molecular oxygen. The sensitivity of the 
luminescence response to ~ a + +  is estimated to extend to as little as a 
small volume of M solution (Shimomura et al., 1963a; Shimomura 
and Johnson, 1973). The light-emitting reaction seems not susceptible to 
inhibition by any compounds commonly occurring in biological systems. 
Moreover, no evidence has been found that aequorin has any toxic prop- 
erties. The speed of response to Ca++ is fast; maximum intensity is reached 
in a few milliseconds (Hastings et al., 1969; Loschen and Chance, 1971). 
https://ntrs.nasa.gov/search.jsp?R=19760008460 2020-03-22T18:05:52+00:00Z
On the basis of sensitivity together with the favorable properties just 
mentioned and a seemingly satisfactory specificity, the use of aequorin 
luminescence as a means of the microdetermination of Ca++ in biological 
systems was suggested (Shimomura et al., 1963b). The method was soon 
applied to  advantage in the detection of changes in Ca++ concentration 
during the contraction of single muscle fibers (Ridgway and Ashley, 1967; 
Ashley and Ridgway, 1968), and, in a number of other physiological pro- 
cesses including metabolic activity of mitochondria (Azzi and Chance, 
1969), activity of single neurons as well as action of inhibitors thereon 
(Baker et al., 1971), and rhythmic discharge of single ganghonic cells 
(Chang et al., 1974). In studies of Ca++ inside single cells, one would be 
most likely dealing with 1 0-17 to 10-I mole ions of ionic calcium, assum- 
ing a cell size of 100 pm in diameter. 
In regard to specificity, extended research on the influence of various 
cations (Izutsu et al., 1972; Shimomura and Johnson, 1973) have shown 
that, under certain conditions of concentration and pH, cations other than 
Ca++ and Srf+ can trigger the luminescence of aequorin. Such cations in- 
clude those of the rare earth elements and also Pb++, Cd++, Co++, and Cuf+. 
In the present study, we tried to improve further the specificity of aequorin 
reaction of Ca++ by masking these cations other than Ca++ through the use 
of various chelating agents. 
MATERIALS AND METHODS 
Purified aequorin of approximately 95 percent purity (Shimomura and 
Johnson, 1969; Johnson and Shimomura, 1972) was desalted by a small 
column of Sephadex G-25 equilibrated with 0.1 rnM ethylene diaminetetra 
acetate (EDTA), pH 7.5. The solution was kept frozen until ready for use. 
CH,COONa, Pb(CH,COO), , CuO, COO, Cd(HC00)2, LaCl,, SrC12 (all 
ultrapure grade) and Y203 (99.999 percent) were obtained from Alpha 
Inorganics, and all other chemicals were reagent grade, with care taken to 
choose those containing a minimum amount of Ca. Deionized distilled 
water having a resistance of more than 10 Mi2 was used, and all solutions 
were prepared and kept in polypropylene containers from which they were 
dispensed with plastic pipets and were not allowed contact with glass through- 
out the experiments, except for dissolving the metal oxides with-HC1 in 
Vycor test tubes as previously described (Shimomura and Johnson, 1973). 
The luminescence reaction was initiated by rapid addition of 4 rnl of 
buffer solution containing the metal salt to 5 pl of aequorin solution con- 
taining 10 pg of aequorin placed in a polycarbonate test tube. Light 
(A max 470 nm) was measured by a photomultiplier-amplifier-recorder 
assembly with a pen response time of approximately 20 ms for the full 
scale. 
RESULTS 
Among various chelating agents tested, sodium diethyldithiocarbamate 
(DDC) was found to be far more effective than 8-hydroxyquinoline, 8- 
hydroxyquinoline-5-sulfonic acid, salycylic acid, oxalic acid, or thiourea, 
in regard to  the masking of Pb++, Cd++, Co++, and Cu++ to improve 
specificity of the aequorin reaction to  Ca++. The data for DDC, summarized 
in table 1, indicate that 1 mM of DDC completely suppressed the activities 
of 0.1 mM each of Pb++, CD++, Co++, and Cu++, but had little effect on the 
activities of Cau, Sr++, and the representatives of the rare earth metals, 
La+* and Y*. Trials with a higher concentration of DDC (5 mM) resulted 
in a considerable decrease in the total light emitted with Cau, indicating 
the partial destruction of aequorin; thus, approximately 1 mM is considered 
to be the optimum concentration for DDC. 
In the absence of DDC, the effects of heavy metal ions are often enhanced 
in the presence of thiol (SH) compounds. For example, Pbf+ and Cd", 
each at a concentration of M in a buffer solution (pH 8) containing 
1 mM of cysteine or 1 mM of 2-mercaptoethanol, both induced a lumines- 
cence of 1.5 X 1012 photonsls. Such luminescence, however, could be 
completely suppressed by addition of 1 mM of DDC, the same as without 
SH compounds as shown in table 1. 
The activities of 0.1 mM of La+* and Y+++ were suppressed by 8- 
hydroxyquinoline-5-sulfonic acid (1 mM) to a level lower than the activity 
of Sr++, although this reagent was found to be relatively ineffective in 
masking Pb ++ and Cd++ . 
In regard to the microdetermination of Ca++ by the luminescence of aequorin, 
in general this method evidently has some unique advantages from the points 
of view of speed and of harmlessness to biological materials. Furthermore, 
it is satisfactorily specific to Ca* at a pH of 8 in the absence of rare earth 
elements and of high concentrations of Sr++. Although the activity of 
Pb++, Co++, Cu++, and Cdu can be completely eliminated by adding DDC, 
tl;e occurrence of these ions in biological systems is normally unlikely; if 
DDC is used, however, control tests for possible harmful effects are ob- 
viously required. 
ACKNOWLEDGMENT 
We thank the National Science Foundation and the Office of Naval 
Research for partial support of this research. Reproduction in whole or 
in part for any purpose of the United States Government is permitted. 
Table 1 
Influence of Sodium Diethyldithiocarbamate @DC) on the Maximum Intensi- 
ties* of Aequorint Luminescence Induced by Various Metal Ions$ 
* Expressed in 1012 photonsls. At 298 K (25'C). 
t 10 fig aequorin (potentiality = 42 X 1012 photons) was used in each test. 
Metal ion 
added 
(0.1 mM) 
None 
Ca++ 
Sr++ 
pb++ 
CO++ 
Cu++ 
Cd++ 
y+++ 
~ a + + +  
$ In the present tests, those cations which have been found inactive fn 
eliciting a luminescence reaction of aequorin, namely, K' ~ e * ,  M~*, 
~ a * ,  ~ n * ,  ~ i * ,  ~ e * ,  ~e*, and ~ n *  (Shimomura and Johnson, 
1973) were omitted. 
In 0.0 1 M glycylglycine-NaOH 
pH 8.0 
8 A momentary flash took place at the start of the reaction (1.5 X 1012 
photons in 0.1 to 0.2 s). 
Without 
DDC 
< 0.02 
40. 
9. 
1.8 
1.8 
0.5 
0.05 
14. 
20. 
In 0.01 M Na-acetate 
pH 6.0 
With 
1.0 mM DDC 
<0.02 
26. 
7. 
< 0.02 
< 0.02 
< 0.02 
< 0.02 
12. 
20. 
Without 
DDC 
<0.02 
36. 
4. 
15. 
0.05 
0.05 
18. 
34. 
24. 
With 
1.0mMDDC 
<0.02 
32. 
4. 
< 0.02 
<0.02 
< 0.02 
< 0.02 
28. 
20. 
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Specificity of aequorin luminescence to calcium

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