1H nuclear magnetic resonance assay of erythrocyte triosephosphate isomerase

ANALYTICAL

BIOCHEMISTRY

197,

178-181

(1991)

‘H Nuclear Magnetic Resonance Assay of Erythrocyte Triosephosphate Isomerase’ Serena

J. Hyslop,

Patricia Beal,* and Philip W. Kuchel’ Department of Biochemistry, University of Sydney, New South Wales2006, Australia; and *The Royal Alexandra Hospital for Children, Camperdown,Sydney, New South Wales2050, Australia

Received

March

4,199l

A direct method for measuring the activity of erythrocyte triosephosphate isomerase using ‘H NMR spectroscopy was developed. NMR spectroscopy allows the simultaneous monitoring of the substrate and the product of the reaction by virtue of the differences in the NMR spectrum of each chemical species. The assay conditions were based on a modification of a conventional .spectrophotometric method. The enzymatic activity measured using NMR gave results comparable to those obtained in a standard assay. The results were used in the kinetic characterization of triosephosphate isomerase in hemolysates from subjects with homozygous or heterozygous deficiency of the enzyme. In general, NMR spectroscopy has the potential for wide application in the rapid development of new enzyme assays. 0 1991

Academic

Press,

Inc.

Triosephosphate isomerase (EC 5.3.1.1; D-glyceraldehyde-3-phosphate ketol-isomerase) is the glycolytic enzyme that catalyzes the reversible isomerization of dihydroxyacetone phosphate (GrnP)3 to glyceraldehyde 3-phosphate (GraP). Erythrocyte enzyme activities have been conventionally measured by spectrophotometric methods (1). The majority of these assays rely on coupled enzyme systems involving the reduction of NAD(P). Here we describe a direct method for measuring triosephosphate isomerase activity using ‘H NMR spectroscopy based on a modification of the buffer con-

r This work was supported by a grant from the Australian National Health and Medical Research Council. S.J.H. gratefully acknowledges the support of a University of Sydney Consolidated Medical Fund PhD Scholarship. ’ To whom correspondence should be addressed. 3 Abbreviations used: DMSO, dimethyl sulfoxide; GraP, glyceraldehyde 3-phosphate; GrnP, dihydroxyacetone phosphate; OD, optical density.

ditions of a “standard” spectrophotometric method (1). NMR spectroscopy allows the simultaneous monitoring of the substrate(s) and product(s) of many biochemical reactions by virtue of the differences in the NMR spectrum of each chemical species (e.g., (2)). This general feature of the NMR spectra of many reaction systems may be useful for keeping track of the conservation of mass of reactants. Thus possible side reactions, which may influence the estimated velocity of the primary reaction, may be detected either by the emergence of other peaks in the spectrum or by an apparent loss of mass (peak intensity) of the primary reactants. The present NMR method was established as an alternative to the conventional procedures, with the view to assisting in the kinetic characterization of erythrocytes from subjects with homozygous or heterozygous triosephosphate isomerase deficiency. Triosephosphate isomerase deficiency is a severe autosomal recessive disorder characterized by a multisystern disease (3-5). The disease is clinically manifested as moderately severe nonspherocytic hemolytic anemia, progressive neurological dysfunction (6), an increased susceptibility to infection (4), and a tendency for sudden cardiac death (78). Deficiency of the enzyme appears to involve all the tissues of the body (4,5). Red cells of severely deficient subjects characteristically possess 5 to 20% of normal triosephosphate isomerase activity (9). MATERIALS

AND

METHODS

Materials. DL-Glyceraldehyde 3-phosphate, imidazole, microcrystalline cellulose (Sigmacell Type 50), and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO). Dr.,-Glyceraldehyde 3phosphate-diethylacetal monobarium salt, NADH, Tris-HCl, triosephosphate isomerase (EC 5.3.1.1; Dglyceraldehyde-3-phosphate ketol-isomerase), and (Yglycerophosphate dehydrogenase (EC 1.1.1.8; glycerol0003-2697/91$3.00

178 All

Copyright 0 1991 rights of reproduction

by Academic Press, Inc. in any form reserved.

‘H

NMR

ASSAY

OF

TRIOSEPHOSPHATE

3-phosphate dehydrogenase) were obtained from Boehringer-Mannheim Australia (North Ryde, NSW, Australia). cY-Cellulose was from Whatman Inc. (Maidstone, Kent, England). 2H20 (99.75%) was from the Australian Institute of Nuclear Science and Engineering (Lucas Heights, NSW, Australia). Heparin (25000 IU/ml) was from Weddel Pharmaceuticals (Sydney, NSW, Australia). Cellent (CE-310L), Histan (HS-lo), and Quicklyser (QLY-BOOA) were obtained from Toa Medical Electronics Co. Ltd. (Kobe, Japan). All other reagents were of analytical reagent grade. Hemolysatepreparation. The method of preparation closely followed that described by Beutler (1). Venous blood was collected into heparinized tubes (100 IU/lO ml whole blood), mixed 1:l with isotonic saline, and filtered through a microcrystalline cellulose:cY-cellulose (1:l) column preequilibrated with isotonic saline. The red cells were eluted with isotonic saline and repacked by centrifugation (3000g); the plasma/saline supernatant was removed by aspiration. Hemolysates to be used in the NMR assay were prepared initially by a 1:2 dilution with Beutler’s stabilizing solution (2.7 mM EDTA, 0.7 mM mercaptoethanol) (1) and then stored at -20°C. Further dilution was carried out as required, prior to assaying. Hemolysates to be used in the spectrophotometric assay were diluted 1:20 with stabilizing solution and used either fresh or after overnight storage at 4°C. Hemoglobin estimations were conducted either on a Sysmex Microcellcounter CC-130 (Toa Medical Electronics Co. Ltd., Kobe, Japan) using the Histan method or manually using the method of Van Kampen and Zijlstra (10). Aquisition of NMR spectra. All spectra were acquired on a Bruker AMX 400 NMR spectrometer operating in the Fourier transform mode. To minimize the time required for thermal equilibrium of the sample and thus stability of the field-frequency lock signal prior to spectral acquisition, the assay was conducted at 30°C. The sample temperature was “set” to 30°C but the actual value was determined by the method of Bubb et al. (11); the mean and standard deviation of the temperatures of assays in the present study were 30.3 +- 0.4”C. Fully nuclear-relaxed spectra were acquired using a standard single-pulse sequence with saturation of the water resonance during the preacquisition delay of 20.17 s; the total recycle time was 23.17 s (five times the longest T,, spin-lattice, relaxation time). T1 measurements were made using a composite 180“ pulse sequence (12). Spectral data were averaged into 8K memory locations over a spectral width of 4.5 kHz. The signal from 2H20 was used for field-frequency locking. Quantitative analysis was made possible by routine incorporation of a known concentration of DMSO (1 mM) in the sample as an internal peak intensity and chemical shift (6, 2.511 ppm) reference.

ISOMERASE

179

NMR time courses. The reaction was initiated by the addition of an aliquot of diluted hemolysate to the NMR assay mixture consisting of -10 m&f DL-GraP, 1 mM DMSO, and 100 mM imidazole buffer, pH* 7.6, constituted in 2H,0 (pH* denotes uncorrected pH meter reading). Six-hundred-microliter sample volumes were used in ‘H NMR experiments. NMR spectral acquisition routinely commenced within 3 min of initiation of the reaction. Spectra were acquired continuously for -25 min. Each spectrum took -3 min to acquire and represented the sum of eight transients. Spectrophotometric time courses. Spectrophotometric assays were carried out using l-ml volumes in
RESULTS

AND

DISCUSSION

Figure 1 shows a time course of ‘H NMR spectra obtained when an aliquot of dilute hemolysate was incubated with the NMR assay mixture; the assignments of ‘H NMR resonances to GraP, GrnP, and DMSO are also shown. Assignment of the GrnP resonances was according to the literature (14); GraP assignment was based on previous work within this laboratory (unpublished). The hydrate form of GraP is the major species in solution (29:l); but the aldehyde form is the substrate for triosephosphate isomerase (15). The Tl’s of GraP,(L) (h, hydrate) and DMSO were determined to be 3.01 +- 0.05 s and 4.63 + 0.02 s, respectively. Triosephos-

180

HYSLOP,

BEAL,

AND

GmPk

KUCHEL

Graph

GmP,

DMSO

Crap,

.3 min

I

I

I

4.5

4.0

3.5

I

I

3.0

2.5

Chemical Shift (6, ppm) FIG. 1. added to indicated ments of

Time course of 400-MHz ‘H NMR spectra, showing the region 580 ~1 of NMR triosephosphate isomerase assay mixture (see at the right of each spectrum. Temperature was 30.9”C. NMR resonances to GraP, GrnP (a, aldehyde; h, hydrate; k, ketone),

phate isomerase activity was measured by following the decline in the GraP,(I) doublet as GraP was isomerized to GrnP. This GraP resonance was chosen for analysis because it was clear of other resonances; however, the same results were obtained by following the increase in the GrnP,(3A3B) and GrnP,(~A~B) doublets (results not shown). Figure 2 shows the NMR progress curve derived from the spectra in Fig. 1 (see Materials and Methods).

2.4 to 5.2 ppm, obtained when 20 ~1 of diluted (1:40) hemolysate was Materials and Methods). The time after addition of hemolysate is parameters were as described under Materials and Methods. Assignand DMSO are shown.

Varying the DL-GraP concentration from 11.3 to 14.2 showed that within this range of concentrations the initial reaction velocity is independent of the initial GraP concentration (Table 1). Statistical analysis using Student’s t test (analysis of means) and Snedecor’s F test (equality of variances) (13) showed that the difference between two sets of initial rates (at GraP concentrations of 11.3 and 14.2 InM) was not statisticallv sianificant at P > 0.05. The literature value for the I?,, of mM

TABLE The Effect Hemolysate Triose-phosphate

Assay conditions Normal control assayb Increased GraP concentration in assaf Increased hemolysate concentration in assap Time

(min)

FIG. 2. NMR progress curve derived from the spectra in Fig. 1. The enzyme activity was calculated as described under Materials and Methods. (+) Point not used in the linear least-squares regression.

1

of Increased GraP Concentration and Increased Concentration on the Initial Velocity of the Isomerase Reaction Measured at 30°C

Initial velocity (pmollmin * ml)

Corresponding calculated triosephosphate isomerase activity (IWgHb)

0.159 f 0.009

2350

f 130

0.167 -c 0.004

2470

f

k 0.006

2230

+ 60

0.227

a Errors are standard deviations and values (see Materials and Methods). * 10 ~1 1:30 hemolysate, 11.3 mM GraP. ’ 14.2 mM GraP. d 15 pl of 1:30 hemolysate.

are corrected

60

for 37’C

‘H

NMR

ASSAY

OF

TRIOSEPHOSPHATE

181

ISOMERASE

is substantially more than that of a conventional ultraviolet spectrophotometer and related equipment, NMR spectroscopy has the potential for application in the rapid development of new enzyme assays because of the directness of the detection process for analytes. The direct monitoring of the substrate(s) and product(s) of the reaction of interest obviates time-consuming and costly development of coupled enzyme assay systems. ACKNOWLEDGMENTS O/ 0

loo0

Spectrophotometric

2om Method

3000 (IU/gHb)

FIG. 3. Correlation between triosephosphate isomerase activity measured by the NMR assay and that measured spectrophotometritally for a number of control samples (A), heterozygous (B), and homozygous (C) triosephosphate isomerase-deficient samples. The error bars denote one standard deviation in the estimate of the activity.

Dr. W. A. Bubb and Dr. B. E. Chapman are thanked for assistance with the NMR spectroscopy and Mr. W. G. Lowe is thanked for technical assistance.

REFERENCES 1. Beutler, E. (1984) Red Cell Metabolism: cal Methods, 3rd ed., Grune & Stratton, 2. Kuchel, P. W. (1989) in Analytical NMR hell, S., Eds.), p. 157, Wiley, Chichester. 3. Schneider, Jr. (1965)

GraP is 0.434 mM (16). The NMR method uses a DGraP concentration that is greater than 10 X K, and hence the initial velocity is greater than 91% of V,,. This contrasts with the spectrophotometric method, where the reaction rate is strongly dependent upon the concentration of D-GraP (3 mM = 6.9 X Km, 87% V,, (1)). Furthermore, under the conditions of the NMR assay, the initial reaction velocity was shown to be directly proportional to the hemolysate concentration when this was changed by a factor of 1.5 (Table 1). Triosephosphate isomerase activity measured for a number of normal controls (A) and heterozygous (B) and homozygous (C) deficient subjects (Fig. 3) shows an excellent correlation between the NMR and spectrophotometric methods. The distinction that can be seen between the control subjects and those with either a heterozygous or homozygous triosephosphate isomerase deficiency indicates the suitability of the NMR method for the diagnosis and kinetic characterization of this deficiency in human erythrocytes. In conclusion, the NMR method presented is a simple and direct method for assaying triosephosphate isomerase activity that is both less labor intensive and less expensive in the cost of reagents than previously published methods. Although it must be acknowledged that the capital cost of a high-resolution NMR spectrometer

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