NOTES
AND
COMMENTS
GLUCOSE DETERMINATION
With few exceptions present analytical
techniques are not sensitive enough for the
direct assay of specific organic compounds
in seawater. The low concentrations
of
most organic materials and the large
amounts of salt create many difficulties for
the analyst. Enzymatic assays are useful
because of their high specificity, but usually lack the required sensitivity. We have
been able to increase the sensitivity of an
enzymatic assay for glucose and use it for
the direct determination of glucose in natural waters at the level of 1O-8 hc without
prior concentration or extraction.
Glucose is coupled to a reaction that produces stoichiometric
amounts of reduced
pyridine nucleotide coenzyme.
1. Glucose
hexokinase
+ ATP
Mg’+
2. Glucose-6-phosphate + NADP
> Glucose-6-phosphate + ADP.
Glucose-G-P
dehydrogenase
’
6-phospho-
gluconate
+ NADPH
The abbreviations used are: ATP, adenosine triphosphate; ADP, adenosine diphosphate; NADP and NADPH, oxidized and
reduced forms of the coenzyme, nicotinamide adenine dinucleotide phosphate.
The reduced coenzyme is then made to
reduce a dye, resazurin, to a highly fluorescent product, resarufin. This- reaction is
catalyzed by diaphorase.
3. KADPH
+ Resazurin
w
diaphorase
NADP
+ Resarufin.
The amount of resarufin produced is proportional to the amount of glucose present
in the original sample. The use of the dye
for this purpose was described by Guilbault
and Kramer (1964). The reduced coenzyme
is itself fluorescent, but in this system the
1 Contribution
No. 2037 from the Woods Hole
Oceanographic
Institution.
This work was performed under National Science Foundation
Grants
GB 5199 and GB 861.
IN NATURAL
WATERS'
fluorescence produced by the dye in the
final sample is about 20~ that produced
by the same amount of reduced coenzyme.
Rabbit muscle hexokinase (140 units/mg)
dehydrogenase
and glucose-6-phosphate
(140 units/mg) were obtained (Boehringer
Mannheim Corp., 20 Vesey Street, New
York). One unit of enzyme catalyzes the
transformation of a micromole of substrate
per minute at 2%. The enzymes were
stored as (NH&SO4
suspensions at OC.
The diaphorase was a crude preparation
from Clostridium kluyveri (Sigma Chemical
Co., St. Louis, MO., and Worthington Chemical Co., Redbank, N.J.). This enzyme was
centrifuged to remove precipitated material
and kept frozen at -20C. Some diaphorase
preparations contained glucose, which could
be removed by passing 10 ml of enzyme
through a column ( IO-cm long, 5-cm diam )
of Sephadex G 100 equilibrated with 0.05 M
Tris buffer pH 8.0 and collecting only the
first yellow, protein-containing
band eluted
from the column. Resazurin was a gift from
Dr. Guilbault. Commercial preparations of
resazurin varied widely in blank value. The
resazurin was dissolved in a small amount
of methyl cellosolve and diluted in water
to prepare a 0.1 rnnf stock solution. Stock
solutions of 10 mM NADP, 100 mM ATP
(pH 7), 10 mg/ml bovine serum albumin,
0.1 mM resazurin, and the 0.200 M glucose
standard were stored at -20C.
Freshly collected water samples were filtered through 45mm-diam
(0.45-p pore
size) Millipore filters previously washed by
soaking in distilled water for several hours.
The filtrate was collected in a flask submerged in an ice bath. Gentle filtration was
used to avoid breaking plankton cells on
the filter. Filtration by gravity with a pressure head of less than 10 cm of water can
be used for samples smaller than 250 ml.
A glucose reagent was prepared for each
analysis. For freshwater samples, it contained 0.2 M Tris(hydroxymethyl)aminomethane pH 7.8, 0.01 mM NADP, 1.0 rnxt
361
--
362
NOTES
AND
ATP, 10 rnhf MgCl?, 0.2 mg/ml bovine
serum albumin, 0.4 units/ml of hexokinase,
and 0.2 units/ml of glucose-6-phosphate dehydrogenase. The enzymes were added just
before use.
Adjustments must be made to this reagent for use in seawater samples. The high
salt concentrations decrease the substrate
affinity and thus lower the effective activity of the enzymes. In addition, the 0.01 M
S04”- present in seawater inhibits glucose6-phosphate dehydrogenase. Therefore, the
NADP concentration was increased to 0.1
mM and the enzyme levels doubled, permitting the reactions to take place at salt
concentrations as high as 0.25 N, but at a
low rate. Full strength seawater was diluted with 2 volumes of distilled water to
reduce the salt concentration to about 0.15
N and increase the reaction rate. Dilution
to 0.15 N was chosen as a compromise between loss of sensitivity and increased reaction time.
Volumes of 300 ~1 of glucose reagent
were added to 3.00-ml samples of freshwater or diluted seawater in 12- x 75mm
culture tubes and mixed by inverting several times. Freshwater samples were incubated for 20 min and seawater samples
for 90-120 min at 25C. A mixture of one
part 0.1 mM resazurin to two parts 50 units/
ml diaphorase was prepared and 30 J of
this was added to each tube. The tubes
were inverted as before and the fluorescence of each sample measured after a few
minutes.
In such analyses, the blank is of great
importance. Since it could not be assumed
that any sample of water was completely
free of glucose, two types of blanks were
used. In the first type, one of the essential
reagents, ATP or an enzyme, was omitted
from the glucose reagent. In the second
type, the blank contained a complete glucose reagent and was incubated with the experimental samples through the first steps.
Then the blank was treated with HCl to
about pH 2 (60 ~1 of 1 N HCI) and all the
tubes were heated at 60C for 15 min.
NADPH is very unstable in acid and this
mild treatment is sufficient to destroy all
COMMENT
NADPH formed from any glucose that
might have been present in the blank
(Lowry, Passonean, and Rock 1961). Heating did not affect the NADPH in the other
tubes which were still at ;pH 7.8. The blank
was neutralized with NaOH (60 ~1 of 1 K
NaOH ) and the experimental samples and
standards received an equivalent amount
of NaCl (120 ~1 of 0.5 N NaCl) before the
addition of resazurin and diaphorase. The
fluorescence of these two types of blanks
was the same.
Available commercial enzyme preparations are specific and free of interfering
contaminants. Hexokinase will phosphorylate hexoses in the order glucose > fructose > mannose, but glucose-6-phosphate
dehydrogenase is highly specific for both
substrate and coenzyme. The agreement
between blank values obtained by these
two methods and the high specificity of
the dehydrogenase enzyme give confidence
in the method.
Fluorometric
measurements were made
in a fluorometer (Turner model 110 or 111)
equipped with a green phosphor lamp
and a temperature-stabilized
reflecting cell
holder. Samples were kept at a uniform
since fluorescence
changes
temperature
with temperature. Interference filters (Baird
Atomic B-10) with transmission peaks at
520 rnp and 580 rnp were used as primary
and secondary filters. The excitation and
emission peaks of resarufin are quite close,
so the amount of scattered excitation light
that might pass the secondary filter was
reduced by using filters having peak transmissions on the lower wavelength side of
the excitation peak and on the higher wavelength side of the emission peak. A Polaroid filter was also included on the excitation side and positioned to reduce the polarized light reflected from the walls of the
glass sample tubes. All solutions were kept
free of light-scattering
particles and clean,
unscratched culture tubes were used. With
these precautions it was possible to read
the samples on the highest sensitivity range
of the fluorometer.
All analyses included, routinely and in
triplicate, blanks, the sample of water with
363
NOTES AND COMMENT
no added glucose, and a series of standards
having increasing amounts of glucose added
to the experimental sample of water. Glucose standards were prepared by serial dilutions from a 0.200 M stock solution. The
difference between the sample with no
added glucose and the blank was proportional to the glucose present in the water.
The concentration
could be determined
from the standard curve which was a
straight line over a wide range of concentrations. With the use of constriction micropipettes for small volumes and with careful
attention to pipetting, multiple analyses of
a sample usually agreed within a few fluorimeter units. Because of this reproducibility we have confidence in determinations
where the blank value was as much as 80%
of the experimental value. This sets the
lower limit of sensitivity at about 1 x 10-S
M glucose in freshwater and 3 X 1O-s M in
seawater (which must be diluted three
times before analysis ) .
We have been able to measure the
amount of glucose in local fresh and brack-
ish ponds and in inshore seawater with this
technique. The values are usually in the
range of 2 to 6 X 1O-8 M. A more extensive
series of measurements in the open ocean
is described elsewhere (Vaccaro et al. 1968).
SONJA E. HICKS~
FRANCIS G. CAREY
Woods Hole Oceanographic Institution,
Woods Hole, Massachusetts
02543.
REFERENCES
GUILBAULT, G. G., AND D. N. KRAMER. 1964.
New direct fluorometric
method for measuring dehydrogenase
activity.
Anal.
Chem.,
36: 2497-2498.
LOWRY,
0. H., J. V. PASSONEAN, AND M. K. ROCK.
1961. The stability
of pyridine
nucleotides.
J. Biol. Chem., 10: 2756-2759.
VACCARO, R. F., S. E. HICKS, H. W. JANNASCH,
AND F. G. CAREY. 1968. The occurrence
an d role of glucose in seawater.
Limnol.
Oceanog., 13 : 356-360.
2 Present address:
Massachusetts
Technology,
Department
of Nutrition,
Massachusetts.
Institute
of
Cambridge,
TEMPERATURE ,~ND CURRENT OBSERVATIONS IN CRATER LAKE, OREGON*
Crater Lake occupies a caldera formed
when the ancient Mt. Mazama collapsed
within itself. The near circular lake (shoreline development of 1.33) is 589 m deep
and 55 km2 in area. The lake surface is
presently 1,882 m above mean sea level;
however, the level of the lake has fluctuated as much as 4.5 m in the last 20 years
(Byrne 1965). The extreme clarity of the
water adds to the unique physical characteristics of Crater Lake.
There has been some debate in the past
as to the degree of thermal stratification
that takes place during summer. Hasler
(1938) reported definite temperature stratification during July and August. Fairbanks
(cited in Nelson 1961) also collected data
indicating thermal stratification.
Temperature profiles taken by Kemmerer, Bovard,
l Oregon Agricultural
nical Paper No. 2354.
Experiment
Station
Tech-
and Boorman (1924) led them to believe
that Crater Lake might not stratify thermally.
Another unusual thermal feature of the
lake is that it seldom freezes over. Ice was
observed to cover the lake from midFebruary through April in 1949 (Ruhle
1949), for two days in 1924, and possibly
during the winters of 1887 and 1898
(Waesche 1934).
There is a paucity of data on the currents
of the lake. The only previously known
attempt to study surface currents was by
Kartchner and Doerr (1939) who followed
movements of a vertical floating log.
In an effort to describe further the physical features of Crater Lake, temperature
and current measurements were made in
summer 1966. Vertical and horizontal temperature profiles were taken and drift devices were released to follow the surface
currents .