Table Of ContentA FLEXIBLE SYSTEM
OF ENZYMATIC ANALYSIS
OLIVER H. LOWRY
Department of Pharmacology
Washington University School of Medicine
St. Louis, Missouri
JANET V. PASSONNEAU
National Institute of Neurological
Diseases and Stroke
National Institutes of Health
Bethesda, Maryland
1972
ACADEMIC PRESS New York and London
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PREFACE
This is a multipurpose manual of laboratory methods. It offers a systematic
scheme for the analysis of biological materials from the level of the whole
organ down to the single cell and beyond. It is intended as a guide to the
development of new methods, to the refinement of old ones, and to the
adaptation in general of methods to almost any scale of sensitivity.
The incentives for the methodology in this book came from the rigorous
demands of quantitative histochemistry and cytochemistry. These demands
are specificity, simplicity, flexibility, and, of course, sensitivity—all likewise
desirable attributes of methods for other purposes.
The specificity is provided by the use of enzyme methods. Simplicity is
achieved by leading all reactions to a final pyridine nucleotide step. Flexi-
bility and part of the sensitivity result from the use of fluorometry for meas-
uring the oxidation or reduction of the pyridine nucleotide. Greater sensitivity
is provided by chemical amplification by means of "enzymatic cycling."
The book is in a sense narrow. It does not have the comprehensive
character of either Bergmeyer's " Methods of Enzymatic Analysis" or of
Glick's " Quantitative Histochemistry." Instead, with a few exceptions, only
methods and tools actually used by the authors and their long-suffering
colleagues and trainees are presented. It is hoped, however, that the principles
presented can be exploited for new analyses when needed and therefore
compensate for the lack of breadth.
The book is intended for novices as well as experts who are familiar with
other analytical styles. It contains, therefore, rudimentary information,
perhaps oversimplified, on such matters as enzyme kinetics and the use of
buffers. It is divided into three parts: a general section, one on quantitative
histochemistry, and an appendix containing information that may be useful
to have at the bench.
xi
xii Preface
The general section is comprised of nine chapters, the first three of which
deal with the properties of the pyridine nucleotides, kinetics, and glassware.
Chapter 4 describes in considerable detail the methods for three metabolites.
In each case 5 or 6 protocols, covering a 100,000-fold range of sensitivity,
are given, together with the principles involved in making the sensitivity
changes. Chapter 5 is a similar presentation for three enzymes. Chapter 6,
"Improvement, Modification, Adaptation, Trouble Shooting, and Develop-
ment of New Methods," is meant to be the " heart" of the book. We hope
most users will read this chapter. The remaining chapters in this section deal
with the preparation of tissues for analysis, the enzymatic cycling methods,
and a compendium of thirty-six metabolite assays.
The quantitative histochemistry section is comprised of four chapters
which include information on the preparation of frozen-dried material and
dissection of samples for analysis, the fishpole balance for weighing samples,
and, finally, the generalities of analysis with emphasis on the "oil well
technique."
It will be seen that no presentation is made of methods dependent on
radioactive tracers. There is no implication from this that radioactive methods
are not at least as important as direct chemical methods. On the contrary, we
would argue that one complements the other. One common misconception
worth dispelling is that radioactive methods are inherently more sensitive
than chemical ones. For example, to count carrier-free 14C in 2 hours with an
error of not over 3% requires about 5 x 10"14 moles. Direct chemical assays
with a single enzymatic cycling step can provide 50 times this sensitivity and
with double cycling 50,000 times this sensitivity.
Unless noted otherwise, abbreviations for chemical compounds will follow
the recommendations of the Journal of Biological Chemistry. The symbols
M, mM, μΜ, nM, 10"8 M, etc., will be used exclusively to indicate concen-
tration, i.e. molar, millimolar, micromolar, nanomolar, 10"8 molar, etc.
Amounts of material will be indicated by the terms moles, mmoles, ^moles,
10"12 moles, etc. For example, an enzyme velocity given as 20 μΜ/min would
mean 20 ^moles per liter per minute (20 ^molar/ minute). Wavelengths will be
given as nm (nanometers or ιημ). Enzyme activities will be given in inter-
national units (U), i.e. ^moles/minute.
We wish to acknowledge the ideas and criticism generously given by many
colleagues. Special thanks must go to Drs. Helen B. Burch, David. B. Mc-
Dougal, and Franz M. Matschinsky and to Demoy W. Schulz, Joyce G.
Carter, and Joseph G. Brown, and to a long series of patient postdoctoral
fellows who have struggled with us to develop and use this methodololgy.
Oliver H. Lowry
Janet V. Passonneau
CHAPTER 1
PYRIDINE NUCLEOTIDES
All of the methods of enzymatic analysis to be considered depend on
pyridine nucleotides. In 1935 Negelein and Haas in Warburg's laboratory
described a method for the determination of glucose-6-P dehydrogenase
activity based on the increase in absorption in the near-ultraviolet as TPNH
was produced. This is apparently the first publication of a method of this
type. Greengard (1956) was the first to describe fluorometric pyridine nucleo-
tide methods for measuring metabolites. Since 1935 an enormous number of
enzymes and metabolites have been measured with the aid of DPN and TPN.
In fact, with the help of auxiliary enzymes almost every substance of biologi-
cal interest could be measured with a pyridine nucleotide system.
The usefulness of DPN and TPN for analytical purposes depends on some
of their unusual properties. (1) They serve as the natural oxidizing and reduc-
ing agents in a wide variety of specific enzyme systems. With the appropriate
enzyme as catalyst, they can selectively oxidize or reduce a particular substrate
in the presence of innumerable other compounds. If one or more auxiliary
enzymes are used, it is a rare substance which cannot be specifically oxidized
or reduced in this way. (2) The reduced forms of the nucleotides, DPNH and
TPNH, are fluorescent whereas the oxidized forms are not. Because the
fluorescence can be measured accurately at concentrations down to 10"7 M,
oxidation or reduction of a pyridine nucleotide can be measured with great
sensitivity. (3) The reduced forms can be completely destroyed in acid without
3
4 1. Pyridine Nucleotides
affecting the oxidized forms. Conversely, the oxidized forms can be destroyed
in alkali without affecting the reduced forms. This means that at the end of a
reaction the excess pyridine nucleotide of the reagent can be destroyed,
leaving the fraction that has been oxidized or reduced intact for subsequent
measurement by procedures which provide even greater sensitivity than the
native fluorescence. (4) At this point two possibilities are available. Both the
oxidized and reduced forms can be converted to highly fluorescent forms in
strong alkali. This permits accurate measurements at concentrations down to
10"8 M. Alternatively, much greater sensitivity can be provided by "enzy-
matic cycling" in which the pyridine nucleotide acts as the catalytic inter-
mediate for a two-enzyme system (Chapter 8).
To make the fullest analytical use of the pyridine nucleotides it is desirable
to be thoroughly familiar with those properties which have analytical rele-
vance. In this chapter, these properties will be described, together with a
discussion of fluorometry and its limitations. In addition, the preparation,
standardization and storage of pyridine nucleotides will be detailed.
Spectral Absorption and Fluorescence of Reduced Pyridine Nucleotides
DPNH and TPNH have identical absorption bands in the near-ultra-
violet which peak at 340 nm. DPN+ and TPN+ do not absorb at this wave-
length. Therefore changes in oxidation or reduction can be measured in the
spectrophotometer.
Some of the light which is absorbed is reemitted as fluorescence. Although
only a small fraction of the absorbed light is emitted, this can be measured to
give much greater sensitivity than can be easily attained from measurement of
the absorption itself. In the spectrophotometer (i.e., with absorption measure-
ments) it is the nonabsorbed light that is registered on the phototube, and
very small decreases in this transmitted light are hard to measure. The ordi-
nary spectrophotometer requires for precision a decrease in the transmitted
light of 5 % or more, and there is little to be gained by increasing the intensity
of the incident light or sensitivity of the phototube. In the fluorometer, the
photocell sees only the emitted fluorescence and this is directly proportional
to the intensity of the incident light. Thus, the sensitivity can be increased
almost without limit by increasing the intensity of the light source, the ampli-
fication, or the phototube sensitivity (by using a photomultiplier tube).
Although spectrophotometry does not readily offer the sensitivity of
fluorometry, it is exceedingly useful for standardization purposes and for
measurements with concentrations of DPNH and TPNH above the range
suitable for fluorometry. The molar extinction coefficient (ε) for DPNH or
TPNH at 340 nm is 6270. This means that a 0.1 mM (100 μΜ) solution would
have an optical density (O.D.) of 0.627. This corresponds to 23.6% trans-
Limitations of Fluorometry 5
mission (/) through a 1 cm light path. [With a 1 cm light path, log (I /I) =
0
O.D., or for a 100 μΜ solution, log (100/23.6) = 0.627.]
With ordinary equipment accurate measurements can be made with
DPNH concentrations of 10-200 μΜ (O.D., 0.06-1.2). With volumes of 0.5 ml
this corresponds to 5-100 x 10"9 mole. With special equipment (microcells
or special differential amplifiers) much smaller amounts of DPNH can be
measured. But fluorometry offers a simpler and easier way to achieve high
sensitivity.
In the fluorometer the analytically useful range of concentration with
DPNH (TPNH) is from 0.1 to 10 μΜ, equivalent to 0.1-10 x 10"9 mole in
a 1 ml volume. Thus, the upper limit for fluorometry is about the lower limit
for accurate spectrophotometry (with conventional equipment). Fluoro-
metric sensitivity can be further increased, and extended to oxidized as well as
reduced pyridine nucleotides, by treatment with strong alkali (see below).
Limitations of Fluorometry
For the great advantages of fluorometry a certain price has to be paid. It
has a few peculiarities and limitations which should be clearly recognized.
QUENCHING
Any substance which absorbs the exciting light, including the pyridine
nucleotide itself, will reduce the intensity of the exciting light as it passes
through the solution. This will thereby diminish the emitted light, i.e., will
"quench" the fluorescence. In consequence, the emitted light is proportional
to the concentration of the substance measured only so long as the absorption
of the exciting light is negligible. It is for this reason that the upper concen-
tration limit for DPNH measurement is set at about 10 μΜ (corresponding to
an O.D. of 0.063 or a 5 % absorption of light at the middle of a 1 ml fluoro-
meter tube).
A second source of error is absorption of part of the emitted light by
colored substances which might be present. Fortunately, this is seldom a
problem. Because the fluorescence is in the visible region, the presence of
absorbing materials can be detected by eye.
FLUORESCENCE ENHANCEMENT
A number of cations can enhance the fluorescence of pyridine nucleotides
in alkaline solution. For example, the presence of 1 mM Mg2+ at pH 11.5
will enhance the native fluorescence of DPNH by 50% and that of TPNH by
300%. The enhancement with metals is not analytically useful, since it is
affected by the order of mixing and the fluorescence is not stable with time.
Enhancement by metals should therefore be avoided by either adding an
excess of EDTA or keeping the pH at 10 or below for reading.
6 L Pyridine Nucleotides
EFFECT OF ENZYMES ON NATIVE FLUORESCENCE
The fluorescence of DPNH or TPNH may be affected (usually enhanced)
by the enzymes they serve as coenzyme. In most situations this is not a
problem. It can become significant when high levels of enzyme are used or if
the enzyme has a very strong effect. The difficulty is that the enhancement is
not the same at all coenzyme levels, since only that fraction of the coenzyme
which is in combination with the enzyme is affected. Consequently pro-
portionality is lost at some point, usually at a low level, because high molar
concentrations of enzyme are seldom used. The safe procedure is to test the
system with several levels of DPNH or TPNH in the working range and if
necessary calculate analytical results from a standard curve.
EFFECT OF pH
There appears to be no effect of pH per se on the fluorescence of DPNH
(TPNH) from pH 6 to pH 13. However pH's below 7 are ordinarily avoided
because of instability of the reduced forms. At pH's above 10.5, DPN+
(TPN+) begins to be converted into a fluorescent product. Therefore, high pH
is avoided when the oxidized nucleotides are present.
TEMPERATURE EFFECTS
Another potential source of error is the large negative temperature
coefficient which is characteristic of fluorescence in general. In the case of
pyridine nucleotides this amounts to 1.6% per degree. If standards and sam-
ples are all read at the same temperature, there is of course no problem, but
differences in temperature must be avoided.
FLUORESCENCE BLANKS
An ultimate limitation to fluorescence sensitivity is set by blank readings.
Most reagents are either slightly fluorescent or contain fluorescent impurities.
With H 0 alone it is difficult to reduce the instrumental reading below a value
2
equivalent to the fluorescence of 0.05 μΜ DPNH. Part of this is due to failure
of the optical system to exclude all scattered light from the phototube; part
may be due to slight fluorescence of the light filters; and part is possibly due
to the Raman spectrum of the H 0 itself.
2
An increase in scattered light, whether from a scratched tube or from
turbidity in the solution will increase the blank even in the best fluorometer.
In a poorly designed instrument, or with improper filters the effect will be
much greater.
It is unnecessary to tolerate a blank with distilled H 0 greater than the
2
equivalent of 0.1 μΜ DPNH. If the H 0 blank is higher than this the fault
2
can be with the fluorometer, the filters, the H 0, the vessel used to store the
2
H 0, or the fluorometer tubes (scratched, dirty, or dusty).
2
Fluorescence Reference Standards 7
LIMITS OF USEFUL SENSITIVITY
In many instances the useful sensitivity of a fluorescence measurement is
set by factors outside of instrumentation. As sensitivity is increased, contami-
nation becomes an increasing problem. Certain substances such as inorganic
phosphate or ammonia or lactate are so common that it becomes impracti-
cable to measure them at concentrations below 10~6 M. In general, at the
highest sensitivity there is certain to be danger of contamination with traces
of fluorescent material or specks of dust. The greatest useful direct sensitivity
is achievable if the assay is based on the difference between two readings made
before and after the addition of the specific reagent. It is helpful if the time
lapse is not too great and essential that the instrument be highly stable.
Fluorescence Reference Standards
All fluorescence readings have to be made by comparison with some
reference solution. A stable working standard such as quinine sulfate is
ordinarily used, and all readings made against it. Such standards ought not
to serve as a basis for calculation, since they may change with time and may
not have the same temperature coefficient as DPNH. In each analytical pro-
cedure a true standard is carried through the entire process.
Some problems have been encountered in preparing stable quinine stan-
dards of the concentrations desired. The standards are routinely prepared in
test tubes like those used for fluorometric analyses. If the tubes are sealed with
corks, the solutions evaporate, and require constant rechecking. If the tubes
are sealed by heating in the flame, the quinine solutions frequently do not
fluoresce as much as expected. The following procedure is recommended to
avoid these problems.
The Pyrex tubes (10 x 75 mm) are heated and pulled in an oxygen-gas
flame so that there is a long, narrow neck. At this stage the tubes are
filled with 50% HN0 and heated for 5 or 10 min in a boiling water bath.
3
The tubes are then rinsed thoroughly three or four times with distilled water,
filled with distilled water, and again heated. The water is shaken out and
quinine sulfate (0.002 to 0.2 ^g/ml) prepared in 0.01 N H S0 is added to
2 4
the tubes in a volume of 1 ml. The narrow neck of the tube can then be easily
sealed in the flame without risk to the quinine sulfate solution. Standards
prepared in such a manner yield the expected fluorescence and are relatively
stable with time.
The ratio between the fluorescence readings with quinine and DPNH
varies somewhat with the wavelength of the exciting light and the transmission
characteristics of the secondary light filter. With a tungsten light source and
the secondary filter recommended, quinine is about 30 times more fluores-
scent than DPNH or TPNH on a molar basis. This means that a 1 μΜ DPNH
8 1. Pyridine Nucleotides
solution is equivalent to approximately 0.013 /xg/ml of quinine sulfate.
(Quinine fluorescence is strongly quenced by halide ion, therefore standards
must be prepared in H S0 rather than HCl.)
2 4
Excitation and Emission Wavelengths and Light Filters
The fluorescence spectra of DPNH and TPNH appear to be identical in
position and magnitude in pure solution. Their fluorescence is not light-
sensitive. Because the absorption maximum is 340 nm, this is ordinarily the
optimal wavelength for excitation. There may« be situations, however, in
which the ratio of blank to sample fluorescence can be reduced by using a
somewhat longer wavelength. The usual fluorometer actually provides
excitation with the 365 nm Hg line. This is no disadvantage as far as sensitivity
is concerned, since the useful sensitivity limit is determined by the blank
fluorescence and not by the absolute output of fluorescent light. With a Hg
light source a very satisfactory primary filter is Corning No. 5860, which has
peak transmission (about 25%) at 360 nm. With the weaker ultraviolet light
provided by a tungsten light source Corning filter No. 5840 with peak
transmission (about 70%) at 355 nm may be preferable, especially for weak
solutions.
The fluorescence emission maximum is at 460 nm. Here again the best
wavelength for measurement may not always be at the peak. The actual
choice can be made empirically by finding the optimal ratio between sample
and blank fluorescences. For general purposes the best filter combination we
have found consists of a combination of Corning filters No. 4303 and No.
3387. Because No. 3387 is slightly fluorescent, it is placed nearest the photocell,
where it is shielded from most of the scattered light by the other filters. Other-
wise somewhat higher blank readings will be obtained.
Conversion of Pyridine Nucleotides in Alkali into Fluorescent Products
As discovered by Kaplan et ah (1951), the destruction of DPN+ and
TPN+ in strong alkali results in the formation of highly fluorescent products.
There is no difference between the products from the two nucleotides in
regard to absorption spectrum, fluorescence spectrum, or fluorescence
intensity. DPNH and TPNH, which are very stable in alkali, can also be
converted into these products if they are first oxidized to DPN+ and TPN + .
The absorption spectrum has its peak at 360 nm, i.e., it is shifted only 20
nm from the peak for the native reduced nucleotides. The fluorescence spec-
trum is narrower than in the case of DPNH and TPNH but the peak is in the
same position (460 nm; Lowry et al., 1957). The fluorescence under recom-
mended conditions is about 10 times greater than the native fluorescence of
