Principle and method of measurement.
The
statement of the principle of measurement of the
method should include the techniques of analyte
separation and of signal generation and detection.
The phosphate method of Luque de Castro
et al.
does not employ an analyte separation step. The
signal is generated by an enzymatic reaction for
phosphate coupled to the production of an fluores-
cent end-product,
A number of enzymatic methods . . . are
based on the ability of phosphate to activate
some enzymatic reactions catalyzed by
glyceraldehyde-3-phosphate
dehydrogenase
(8), phosphorylase
a
(9), maltose phosphory-
lase (10), sucrose phosphorylase (11), and
nucleoside phosphorylase
The method of Luque de Castro
et al.
utilizes
nucleoside phosphorylase as the enzyme,
NP
Inosine + P
i
Ö
hypoxanthine + ribose-5-P
XOD
Hypoxanthine + 2 H
2
O
Ö
uric acid + 2 H
2
O
2
HPOX
p-Hydroxyphenylacetic acid +2 H
2
O
2
Ö
H
2
O + bi(p-hydroxyphenylacetic acid)
where NP is nucleoside phosphorylase, XOD is
xanthine oxidase, and HPOX is peroxidase.
The species monitored fluorometrically is the
dimer of
p-
hydroxyphenylacetic acid (
p-
HPA),
which exhibits maximum excitation at 325 nm
and maximum emission at 415 nm.
In this method analyte specificity is achieved both by
the use of an enzymatic reaction specific for
phosphate and by the use of fluorometric detection.
Reagents and apparatus.
As in the written
procedure, the method evaluation report should
stipulate the identity and source of reagents and
describe the preparation of stock solutions and
working solutions. The storage conditions and shelf
lives of the solutions should be stated. The prepara-
tion of the immobilized enzyme reactors (IMERs) is
of particular note in the method of Luque de Castro
et al.
,
NP and XOD were immobilized on
controlled-pore glass (CPG 120-200 mesh;
Electronucleonics, Fairfield, MA) by using
Masoom and Townshend's procedure (17).
Pump tubes of different lengths [1.5 mm
(i.d.)] were then packed with each support-
enzyme conjugate and stored at 4°C in the
following solutions: 100 mmol/L Tris-HCl,
pH 7.0, for the NP immobilized enzyme
reactor (IMER) and 1 mol/L ammonium
sulfate + 0.5 mmol/L sodium salicylate in
100 mmol/L Tris-HCl, pH 7.0, for the XOD
IMER. Under these conditions both enzyme
reactors kept their activity for at least 3
weeks.
The description of the instruments used in the
method should include a detailed discussion of any
modifications required for the performance of the
method. In the case of flow injection systems, such
as that of Luque de Castro
et al
., the flow injection
manifold needs to be described.
The hydrodynamic system used (Fig. 1)
consists of a peristaltic pump that propels the
reagent streams through the channels. The
sample, diluted appropriately, is injected into
a stream of reagent A, which merges with a
stream of reagent B; reagent B contains
inosine, the substrate for NP biocatalysis.
The first two enzymatic reactions take place
along the NP and XOD IMERs. An
additional merging point located after the
IMERs allows the main stream to be mixed
with reagent C (which contains
p-
HPA and
HPOX), which reacts and catalyzes, respec-
tively, the derivatizing reaction of the hydro-
gen peroxide produced in the previous step.
The derivatizing reaction is developed along
the reactor. Finally, the sample reaches the
flow cell and provides the analytical signal.
The enzyme reactor and the open reactor are
thermostated at 37°C.
Preparation and use of the measurement
system.
In most cases, the preparation and use of
the measurement system is a matter of general
laboratory knowledge so no explicit discussion of
these topics is required. If the instrumentation is
new or if a familiar instrument is modified or
operated in a novel fashion, the report should
Laboratory Methods
2-15
