J. T. Baker Chemical. Co., Phillipsburg,. NJ 08865 (lot 724379);. ICN Nutritional. Biochemicals,. Cleveland, OH 44128 (lot 103065); JBL. Chemical Co., San Luis ...
CLIN. CHEM. 29/1, 148-151
(1983)
Effects of Substrate Concentration on Results of Determination of Prostatic Acid Phosphatase with Thymolphthalein Monophosphate Arthur Kessner,2 Eric J. Woodard,3 and George N. Bowers, Jr.’ The relation between concentration of thymolphthalein monophosphate substrate and catalytic activity was in-
vestigated for the determination of prostatic acid phosphatase. This study, an extension of previously reported work (Clin. Chem. 27: 1372, 1981), showsthat lot-to-lot variation in purity of thymolphthalein monophosphate preparations is reflected in substrate-velocity curves. Plateau regions in these curves at 1.5-2.5 gIL result from the combined
effects of (a) substrate concentrations that are an order of magnitude below Km and (b) a further de-
crease in available substrate caused by formation of substrate aggregates in the presence of serum. To simplify the identification of superior lots of thymolphthalein monophosphate, we give a mixed-substrate protocol for testing different lots. AddItional Keyphrases: iting substrate availability error
evidence for a new mechanism limenzyme activity analytical
Thymolphthalein monophosphate (TMP) is a useful substrate for clinical determination of acid phosphatase [ortho-
phosphoric monoester phosphohydrolase (acid optimum), EC 3.1.2.2]. Although this substrate is highly specific for prostatic acid phosphatase (PAP) (1), the measured activity of PAP in serum can vary with the lot of TMP used (2,3). The proposed use of TMP in reference methods for measurement of PAP in serum (4) requires an understanding of this lot-to-lot variability. Other work (2-4) has underscored that the TMP used for PAP measurements must meet strict specifications. Bowers et al. (3) reported on 14 lots of commercially available TMP and established several criteria of acceptabilityforTMP. We have used some of the substrates they described to study their respective substrate-velocity curves in the PAP reaction. Lot-to-lot comparisons were expedited with a protocol involving mixtures of both “test lot” TMP and a “reference lot” of TMP. This mixed-substrate approach allowed identification of minimally inhibiting lots of TMP.
Materials Disodium pliers4 was
thymolphthalein
monophosphate
stored and weighed as previously other reagents were ACS reagent grade.
from
six sup-
described (3). All
Senior Research Student, Trinity
College, Hartford,
P0 Box 1725,Pennsylvania State
CT 06106.
University,
College
of Medicine, Hershey, PA 17033. J. T. Baker Chemical Co., Phillipsburg,
NJ 08865 (lot 724379); ICN Nutritional Biochemicals, Cleveland, OH 44128 (lot 103065); JBL Chemical Co., San Luis Obispo, CA 93401 (lots 68-10-30 and 71-1061); Regis Chemical Co., Morton Grove, IL 60053 (lot P48-115-1B); Serva/Feinbiochemica, Heidelberg, Germany (lot control B from Accurate Chemical Scientific Corp, Hicksville, NY 11801); and Sigma Chemical Co., St. Louis, MO 63178 (lot 47C-02891). Received July 19, 1982; accepted Sept. 13, 1982. 148
disodium TMP, 792 mg of sodium acetate trihydrate, sufficient HC1 to bring the pH to 5.4, and water to volume. It was necessary to warm the material in the flask to completely dissolve certain lots Substrate diluent 5.4; 3.5 gIL Brij-35). (11.7 mL of a 30 g/L 500 mL of de-ionized
Sodium acetate volume brought was adjusted
CLINICAL CHEMISTRY, Vol. 29, No. 1, 1983
to
then diluted to
of TMP. (233 mmol/L sodium acetate buffer, pH Glacial acetic acid (3.12 mL) and Brij-35
solution) were added to approximately water in a 1-L class-A volumetric flask. trihydrate (24.48 g) was then added and the near the mark with de-ionized water. The pH 5.4 with 1.0 mol/L HC1 and the solution was volume.
Alkaline 1.0 moilL
color developer (1.0 moliL sodium carbonate in sodium hydroxide solution). This solution was prepared and used as described elsewhere (2). PAP in pooled human serum. The enzyme preparation
consisted of human seminal fluid in a serum pool at pH 6.5, prepared as previously described (3). This preparation was stored frozen and used freshly thawed.
Methods Estimates of true TMP concentrations from tometric and liquid-chromatographic analyses.
spectropho-
In previous the chemical purity of several lots of TMP was so estimated (3). Those data were used in the present work to calculate “purity indexes.” The following factors were used to correct weighed amounts of substrate for water and impurity content: lot A, (.97); lot B, (.85); lot C, (.80); lot E, (.15); investigations
lot G, (.96); lot I, (.99). Substrate-velocity curves.
We derived substrate concentration-velocity curves according to the assay procedure described elsewhere (2), with the following kept constant: substrate volume = 600 sL; enzyme pool volume 100 giL; assay temperature 29.8 #{176}C; assay time = 60 mm. We calculated =
=
activity in IUB units per liter (U/L) after addition of alkaline color developer using as the molar absorptivity value for thymolphthalein 40 250 L molt cm (3). Effect of reaction time and reaction products. To investi-
gate the
effect
used various
1 Clinical Chemistry Laboratory, Department of Pathology, Hartford Hospital, Hartford, CT 06115. 2 Present address: Beckman Instruments, Inc., Brea, CA 92621.
Present address:
Buffered substrate (2.98 mg/mL TMP Na salt; 233 mrnol/L sodium acetate buffer, pH 5.4; 3.5 gIL Brij-35). A buffered substrate solution was prepared as described by Ewen and Spitzer (2) in a 25-mL volumetric flask, consisting of 10 mL of a 8.75 g/L Brij-35 (detergent) solution, 74.50 ± 0.05 mg of
of reaction
concentrations
time
on
the observed catalysis, we
of reference
buffered
substrate
to measure PAP activity over 5-, 15-, and 60-mm intervals. (The usual volumes of each reaction component were doubled to allow sufficient material for multiple sampling.) Mixed-substrate protocol for substrate evaluation. The usual assay procedure was performed, except that we used mixtures of buffered substrate solutions prepared from different lots of TMP as substrate. To accomplish this, we first pipetted 300 L of the “reference” buffered substrate into the appropriate reaction tubes, followed by an additional 300 fLL of various dilutions of the “test” buffered substrate. Nephelometry of reaction mixtures. Reaction mixtures were prepared containing various concentrations of buffered substrate (0-3.0 mg/mL) plus various amounts of enzyme
consistent with (or contaminant)
noncompetitive or uncompetitive substrate inhibition. However, the presence of congruent plateau regions near 1.0-1.5 mg of TMP per milliliter
in all substrate-velocity curves suggests that additional factors are involved in determining observed PAP activity. To examine this effect, we re-plotted the same data, using TMP molar concentrations corrected for water and impurity content (Figure 1B). The maximal-activity plateaus are now displaced to concentration tested.
-J
>. 0 0
ranges characteristic
for each lot of TMP
A highly pure sample, lot A,5 supported
the highest PAP in the curve corresponding to the region 1.5 to 2.5 mmol/L; this suggests that this may be the true optimal concentration .range for pure TMP. Other lots showed activity,
> C
0 0
with a plateau
diminished
a a
activity in the same concentration
range.
Time-course studies. Substrate-velocity curves for one lot of TMP, lot A, were generated for 5-, 15-, and 60-mm incubations at 29.8 #{176}C. Their shapes are similar despite the various durations of incubation, which suggests that accumulated reaction products do not substantially affect the observed 05
ID
1,5
2.0
25
30
concentration-activity Mixed-substrate
35
Thymolphthaleln Monophosphate (mg/mI)
A. PAP activity (U/L) as a function of substrateconcentration (mg/mL) for four lots of TMP. B. PAPactivity (U/I) as a function of cacuIated IMP concentration (mmol/L) for the samefour lots of IMP. Weighed IMP wascorrected for water and implxity contentusingptzity indexesderivedfrom datapresentedby Bowers et al. (3): index (% IMP by HPLC) X (% TMP by spectrophotometry)/100 [TMPJ 0
4.00
.800
.571
.444
1 #{163}
0.5
these
20
0.4
2.5
D
data can be used to identify superior lots of TMP.
Nephelometry of reaction mixtures. Reaction mixtures containing enzyme pool and buffered substrate at several concentrations were examined by nephelometry at 37 #{176}C. The observed lightscatteringincreased slowly, reaching a lotspecific concentration-dependent levelwithin 5 mm. As little as 10 zL of enzyme pool was sufficient and necessary to allow of aggregates. Larger amounts of the pool caused background light scattering, which obscured the TMP-dependent effects. Figure 4 indicates the effect of substrate concentration on light scattering 5 mm after 10 L of enzyme pool was added to two very different lots of TMP, lot A (the “reference” lot) and lot E (the poorest lot tested to date). thisobserved formation
0.3
g -I>
the uncorrected
C, E, B, and G were “unacceptable” by those criteria. By comparing relative activities at a single “test” concentration,
(rn0VL)
1.33
When
concentration of the “reference” lot was held constant at 1.25 mg/mL (the optimum observed for all lots, see Figure 1A), addition of “test” lots of TMP affected the reaction velocity to an extent characteristic for each lot (Figure 3). For example, lots C and E were more inhibitory than additional lot A (reference), while several lots were similar to or even slightly superior to the reference lot. Note that lots A and I were found “acceptable” by the criteria of Bowers et al. (3), whereas lots
Fig. 1. Effect of substrate concentration on observed PAP activity
-4.00
relation. inhibition studies.
0.2 5.0
0.I
Z IL
10.0
Discussion 0
.500 [TMP)
I.
1.50
2.00
2.50
(i)
Fig. 2. Reciprocal plot of substrate-velocity
data for Lot A
Km can be estimated by extrapolation of the asymptotic portion of the curve
pool. Each mixture was immediately placed into the sample chamber of an automated nephelometer (ICS rate nephelometer; Beckman Instruments, Inc., Brea, CA 92621) in the “scatter” mode, and its light-scattering properties were
monitored.
Results Effect of substrate concentration on PAP activity. The effect of various substrate concentrations (0.265 to 2.55 mg/mL) on observed PAP activity was characteristic for each lot of TMP, variations being particularly marked at the highest substrate concentrations (Figure IA). The presence of minima in the corresponding reciprocal plots (Figure 2) is
Evaluation of various lots of TMP. Although physical and chemical criteria had been developed forevaluatingindividual lotsof TMP (3), a means for understanding the inhibitory propertiesof each lotwas also desirable.Use of the mixedsubstrate protocol provides direct comparisons, which require
fewer test points than are needed to generate complete inhibition curves. Lots identified as “acceptable” and “unacceptable” by Bowers et al. (3) generallyexhibited lessor more inhibition, respectively, when compared with the reference lot at a total substrate concentration of 2.55 mg/mL (Figure 3). Only lot G lacked this correlation, showing less inhibition than lot A despite a previous unacceptable ranking. None of the lotsof substratetestedwas not inhibitory;all lots displayed optimal plateau regions at 1.0to 1.5mg/mL. For “acceptable” lots, this corresponds to 1.5-2.5 mmol of TMP per liter. These experiments provide a means of selecting those Lots A, B, C, E, G, and I correspond, respectively, to lots 14,5,4, 1, 7, and 10 in reference 3.
CLINICALCHEMISTRY, Vol. 29, No. 1, 1983
149
50 0 C >.
#{149} #{149}LotA
40 I>
O-O
0
Lot E
>I-
LU
0
zLU Iz
> LU
30
20
10 THYMOLPHTHALEIN
Fig.
-j
(mg/mi)
MONOPHOSPHATE
3. Mixed-substrate protocol for determining
LU
inhibitory
0
properties of TMP lots Reaction mixture consistsof 1.25mg of reference lot A per milliliter plus 0, 0.25, or 1.25 mg of test lots A (ref), B, C, E, G, or F (lithium salt) per milliliter. Properties of each lot are listed in reference 3
lots from among several “acceptable” candidates that exhibit minimal inhibition of PAP.
Explanation
for Variation
in Substrate
(a) maximum PAP activity and (b) the corresponding strate concentration supporting maximal activity (2,3).
and slope of corresponding
reciprocal
sub-
The plots
(Figure 2) suggest that the PAP-TMP system is characterized by a noncompetitive or uncompetitive inhibition-like pattern. Extrapolation of the asymptotic portion of the curve gave an apparent uninhibited Km value of 10-20 mmol/L, indicating the “optimal” TMP concentration plateau of 1.5-2.5 mmolIL to be about 1/10 Km, or only 1% of the TMP concentration needed to saturate the enzyme. Inhibition studies. All substrates tested, including those previously shown to be 99% pure TMP (3), showed the same pattern of apparent substrate product or contaminant inhibition. Comparisons of substrate-velocity data for one lot at three different reaction times indicate that accumulated product(s) is not a principal cause of decreased activity. Also, inhibition by TMP itself is inconsistent with the observation that lot purity is directly proportional to maximal activity and indirectly related to inhibitory behavior (Figure 1). If strongly inhibitory contaminants were uniquely responsible, those lots with the least inhibitor would show optima at the highest concentration of substrate, whether expressed as weight or molarity. However, Figure 1 indicates that the inhibition is independent of the mass concentration of substrate. Nephelometric studies. All substrate-velocity curves pla-
teaued at similar mass concentrations of substrate; thus it was possible that the “inhibition” might actually be physical substrate depletion caused by aggregate formation of TMP and associated impurities at high concentrations. Probable
contaminants in the substrate include thymolphthalein, and several mono- and di-phosphorylated isomers, species of similar structure and low solubility. The similarity in structure and solubility was evidenced in part by the work of Bowers et al. (3), where similar liquid-chromatographic behavior and similar chromogenicity of the various contaminants were observed. If aggregate formation were to involve nonspecific reactions
of several
such species in the substrate,
then the
plateau would be seen at similar weight concentrations of substrate regardless of the TMP content of the lots investigated. Moreover, the height of each maximal activity plateau 150
0.5
1.0
1.5
2.0
2.5
3.0
(IMP) mg/mL
Fig. 4. Light-scattering properties of buffered substrate in the presence of enzyme pool mixture:500 iL of bufferedsubstrate, 10zL of enzymepool. IMP mass concentration on abscissa. Relative light scatter on ordinate for Lot A and Lot E Reaction
Reactivation
Estimation of Km. Our substrate-velocity data (Figure 1) confirm previously observed lot-dependent variations in both
nonlinearity
0
CLINICAL CHEMISTRY, Vol. 29, No. 1,1983
would depend on the molar TMP concentration available at, the 1.0-2.5 mg/L solubility limit of the substrate. This theory was tested conveniently by using the rate nephelometer, albeit in a mode not usually used for clinical measurements. In the scatter mode, this instrument records light-scattering properties of a specimen in arbitrary units, although itdoes not afford a quantitative the size of the scattering species. Dilutions of two very different lots (A
determination
of
and E) of TMP in
buffered substrate were examined and low constant scatter was observed. However, with addition of 10 L of enzyme pool the results shown in Figure 4 were obtained. The most crucial result
is that
there
was a sharp
break
in the light-scattering
curve. Most interestingly, the mass concentrationof substrate at which thisbreak occurred was identicalfor the reference lot and the impure lot;furthermore, this concentration is
identical to the concentration at the minima of their respective Lineweaver-Burke reciprocal plots. The break in the lightscattering curve may thus represent the crossover point at which larger aggregates are formed. These aggregates apparently involve both serum components and substrate components, because diluted substrate alone, serum alone, or diluent buffer alone shows neither the high levels of scatter nor the break in the curve.
We conclude that it is critical that PAP activity be directly related to the available molar concentration of TMP at all attainable substrate mass concentrations. Errors in estimated TMP concentration because of lot-to-lot variation in purity will be directly reflected in the measured PAP activity. The additional complication of aggregate formation serves to amplify the need more fully to explore kinetic parameters when specific lots of substrate are being selected for clinical use.
References 1. Roy, A. V., Bower, M. E., and Hayden, L. E., Sodium thymolphthalein monophosphate: A new acid phosphatase substrate with greater specificity for the prostatic enzyme in serum. Clin. Chem. 17,
1093-1102 (1971).
2. Ewen, L. M., and Spitzer, R. W., Improved determination of prostatic acid phosphatase(sodium thymolphthalein monophosphate substrate).
Clin. Chem. 22,627-631(1976).
3. Bowers, G. N., Jr., Onoroski, M., Schifreen, R. S.,etal., Spectrophotometric and liquid-chromatographic studies of thymolphthalein monophosphate: Specifications for high-quality substrate for the measurement of prostatic acid phsophatase activity. Clin. Chem. 27, 1372-1377 (1981).
4. Ewen, L. M., Protocol of the manual Reference Method (1980) for acid phosphatase (orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2). Joint NBS and Cooperating Laboratories Enzyme Study Group. NBS Special Publication 260 Series, in preparation.
5. Mangum, B. W., The gallium melting-point standard: Its role in our temperature measuring system. Clin. Chem. 23, 711-718 (1977).
CLIN. CHEM. 29/1, 151-153 (1983)
Column Enzyme Immunoassay for Secretory Immunoglobulin A in Serum Ryohei Yamamoto,1 Shigeki Kimura,’ Shigeko Hattori,’ Yukio Ishiguro,2 and Kanefusa Kato2 This enzyme immunoassay
for specific
secretory immunoglobulin
A concentrations in human
measurement
of
serum involves use of a small chromatographic column as a solid-phase. Serum samples are incubated for 2 h with f3-D-galactosidase-labeled antibody to secretory component, then passed through a 0. 1-mL Sepharose 4B column containing antibodies to human immunoglobulin A. After the column is washed to remove the unbound label, the buffer in the column is replaced by a solution of o-nitrophenyl-/3-D-galactoside
(a /3-D.-galactosidase
substrate)
and incubated at 25 #{176}C overnight. The enzyme reaction is stopped by washing the column with sodium carbonate solution, and the absorbance of the eluate is measured at 420 nm. The concentration of secretory immunoglobulin A can be determined with a minimum detectable sensitivity of 3 mg/L, without interference from free immunoglobulin A and secretory component in the same samples. Additional Keyphrases:
sample preparation
cancer,
chronic infection Secretory immunoglobulin A (SIgA),3 the predominant immunoglobulin found in external secretions, is composed of one molecule each of secretory component (SC) and J-chain, and two molecules of monomeric immunoglobulin A (IgA) (1). SIgA is also present in circulating blood, and concentrations of SIgA (or SC) in serum are reportedly high in patients with carcinomas (2-4) and chronic infectious diseases (5, 6). To clarify the physiological significance of serum SIgA, a simple and specific method for the assay of SIgA is needed. Recently, we have developed a sensitive and specific solid-phase enzyme immunoassay for SIgA with which we could determine the concentrations of SIgA in saliva, sweat, urine, and feces (7). However,
when used with
serum
samples,
the solid-phase
Department of Research and Development, Amano Pharmaceutical Co., Kunotsubo, Nishiharu, Nishikasugai, Aichi 481, Japan. 2 Department of Biochemistry, Institute for Developmental Research, Aichi Prefecture Colony, Kamiya, Kasugai, Aichi 480-03, Japan. Nonstandard abbreviations: IgA, IgG, 1gM, immunoglobulins A, G, and M, respectively; SIgA, secretory IgA; SC, secretory component. Received May 20, 1982; accepted Sept. 1, 1982.
enzyme immunoassay with batch procedure suffers from interference by IgA, present in serum in much greater concentrations than SIgA. We describe here a practical enzyme immunoassay for SIgA in human serum in which interfering IgA is removed by passage through a small column of anti-human IgA antibody immobilized on Sepharose 4B.
Materials and Methods Materials We used de-ionized water throughout. Buffer G. Sodium phosphate buffer (10 mmol/L, pH 7) containing per liter, 0.3 mol of NaCI, 1 mmol of MgCl2, 1 g of bovine serum albumin (Cohn Fraction V; Armour Pharmaceutical Co., Chicago, IL), 5 g of digested gelatin, and 1 g of
NaN3. The digested gelatin was prepared by the treatment of gelatin (Difco Laboratories, Detroit, MI) with protease T1 (Amano Pharmaceutical Co., Nagoya, Japan) as described previously (8). Antigens. SIgA from human
colostrum
(purity
96%)
approx.
was obtained from Behringwerke AG, Marburg, F.R.G., and human IgA and 1gM (substantially free of other immunoglobulins) were from Green Cross Co., Osaka, Japan. Human SC purified from human colostrum according to the method of Kobayashi (9) showed a single band in sodium dodecyl sulfate gel electrophoresis. Concentrations of purified SC, SIgA, IgA, and 1gM were estimated from their respective absorptivities (nm) of 12.7, 13.4, 13.4, and 12.0 (10). Antibodies. Rabbit antiserum to human SC was obtained
from Behringwerke AG, and rabbit antiserum to human IgA (specific to a-chain) was from Medical and Biological Laboratories, Nagoya, Japan. The IgG fractions were isolated from the antisera by precipitation with (NH4)2SO4, dialysis, and chromatography on DEAE-cellulose (11). Anti-IgA antibody immobilized on Sepharose. We mixed IgG fractions of the antiserum to human IgA, 50 mg in 20 mL of sodium carbonate buffer (0.1 mol/L, pH 8.3, containing 0.5
mol of NaC1 per liter), with 10 mL of CNBr-activated
Seph-
arose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden), stirring at 4 #{176}C overnight. The anti-IgA antibody immobilized on Sepharose was then washed with Tris HCI buffer (0.1 mol/L, pH 7.5, containing 0.5 mol of NaCI per liter) and stored
at4#{176}C. (Anti-SC)Fab’-f3-D-galactosidase fragments of anti-SC antibody,
prepared
conjugate. F(ab’)2 by digesting (anti-
CLINICAL CHEMISTRY, Vol. 29, No. 1, 1983
151
