A continuous anaerobic fluorimetric assay for ferrochelataseby monitoring porphyrin disappearance

Zhen Shia and Gloria C. Ferreiraa,b,*

a Department of Biochemistry and Molecular Biology, College of Medicine, Tampa, FL 33612, USAb H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612, USA

Received 9 September 2002

Abstract

A continuous spectrofluorimetric assay for determining ferrochelatase activity has been developed using the physiological

substrates ferrous iron and protoporphyrin IX under strictly anaerobic conditions. In contrast to heme, the product of the fer-

rochelatase-catalyzed reaction, protoporphyrin IX is fluorescent, and therefore the progress of the reaction can be monitored by

following the decrease in protoporphyrin fluorescence intensity (with excitation and emission wavelengths at 505 and 635 nm, re-

spectively). This continuous fluorimetric assay detects activities as low as 0.01 nmol porphyrin consumed min�1, representing an

increase in sensitivity of up to two orders of magnitude over the currently used, discontinuous assays. The determination of the

steady-state kinetic parameters of ferrochelatase yielded KPPIXm ¼ 1:4� 0:2lM, KFe2þ

m ¼ 1:9� 0:3lM, and kcat ¼ 4:0� 0:3 min�1. In

addition to its applicability for acquisition of kinetic data to characterize ferrochelatase and recombinant variants, this new method

should permit detection of low concentrations of ferrochelatase in biological samples.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Ferrochelatase; Continuous assay; Initial rate; Protoporphyrin; Ferrous iron; Anaerobic; Fluorescence; Steady-state kinetics

Ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1),

the terminal enzyme of the heme biosynthetic pathway,

catalyzes the insertionof ferrous iron intoprotoporphyrin

IX to form protoheme. This enzyme has wide phylo-

genetic occurrence and, in most cases, is membrane-

associated (reviewed in [1–3]). In humans, ferrochelatase

deficiency results in erythropoietic protoporphyria [4,5].

Over the past 4 decades several assays have beendeveloped to determine ferrochelatase activity (reviewed

in [3]). One of the most used assays currently for fer-

rochelatase is a discontinuous method, involving ferrous

iron and porphyrin substrates and the spectrophoto-

metric quantification of synthesized heme upon pyri-

dine–hemochromogen formation [6–8]. This assay

detects activities as low as 1 nmol protoheme formed

min�1 [9]. To maintain the iron substrate in the ferrousstate, thiol compounds have been routinely included in

the reaction mixture [8]. However, hemes degrade in the

presence of thiol compounds [8]; additionally, dith-

iothreitol competes with ferrochelatase for binding to

free ferrous iron [10]. A second assay measures fer-

rochelatase activity by following the disappearance of

the porphyrin substrate [8,11–13]. Because under these

assay conditions, the absorption spectra of porphyrin

and heme exhibit clear isosbestic points, indicating

conversion of substrate into product [8], the progress ofthe reaction can be followed using electronic absorption

spectroscopy. While the dual-wavelength assay offered

the prospect of development of a continuous assay, its

inherently reduced sensitivity [8], compared to that of

the pyridine–hemochromogen method, has restricted its

application. In addition, reducing reagents, such as

glutathione, which appear to interfere with heme sta-

bility [8], were included in this procedure [8]. A radio-chemical assay for ferrochelatase activity quantifies the

incorporation of 59Fe into porphyrin [14]. However, this

method has had limited use because of requirements for

safety precautions, expense, and labor [14–16].

To overcome the technical constraints of maintain-

ing the iron substrate in the reduced state and the

Analytical Biochemistry 318 (2003) 18–24

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ANALYTICAL

BIOCHEMISTRY

* Corresponding author. Fax: 813-974-0504.

E-mail address: gferreir@hsc.usf.edu (G.C. Ferreira).

0003-2697/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0003-2697(03)00175-1

requirement of strict anaerobicity, alternative assaystook advantage of the fact that ferrochelatase can cat-

alyze incorporation of other divalent metal ions [12,

13,17–20]. Indeed, a convenient assay is to determine

zinc-chelatase activity [17], since zinc–protoporphyrin

synthesis can be followed continuously by fluorescence

[13,21]. Chelation of Co2þ has also been used to deter-

mine the enzymatic activity of ferrochelatase [12,18,19].

Cobalt-chelatase activity can be measured by the pyri-dine–hemochromogen assay or by monitoring the ab-

sorbance decrease of the porphyrin substrate [12,22,23].

Despite the convenience of these assays using alter-

native metal substrates, kinetic and X-ray absorption

studies [13,24] have provided evidence that chelation of

non-physiological, metal substrates by ferrochelatase

might not be analogous to that of Fe2þ. Camadro and

Labbe [13] demonstrated that while Fe2þ was a com-petitive inhibitor of zinc-chelatase activity with a Ki

value of 50 nM, Zn2þ, although a much less potent in-

hibitor, was a competitive inhibitor for iron chelation

ðKi ¼ 1:5lMÞ. Moreover, the recent X-ray absorption

spectroscopy studies of metal binding sites of mouse and

yeast ferrochelatases indicate that coordination of Zn2þ

is different from that of Fe2þ or Co2þ [24].

Thus, while there are available, convenient methodsto determine the enzymatic activity of ferrochelatase,

each has limitations that reduce its usefulness, particu-

larly in enzyme kinetic studies. The development of a

continuous and sensitive assay for ferrochelatase activity

using the physiological substrates is a necessary pre-

requisite for the steady-state kinetic and mechanistic

characterizations of the enzyme. The implementation of

such a continuous method has encountered several ob-stacles. Major problems have been maintaining the

substrate iron in the reduced state [6,8,12], the relative

insolubility of the porphyrin substrate in aqueous solu-

tion [6,8], the low levels of enzymatic activity

[9,12,25,26], and the small amounts of ferrochelatase in

biological samples. We report here a continuous, fluo-

rimetric assay for ferrochelatase activity, which is con-

ducted under strictly anaerobic conditions to maintainthe iron substrate in the ferrous state and measures the

rate of protoporphyrin IX consumption. Significantly,

by minimizing the inner filter effect that can be associ-

ated with porphyrin fluorescence, the sensitivity of this

method was enhanced up to two orders of magnitude

greater than currently used assays, making it possible to

determine initial enzymatic rates at lower substrate

concentrations than those previously reported.

Materials and methods

Materials. Protoporphyrin IX and N-methylproto-

porphyrin were purchased from Frontier Scientific.

Ferrous ammonium sulfate and sodium citrate were from

Fisher Scientific. Tris(hydroxymethyl)aminomethanecarbonate (Trizma base), polyethylene glycol sorbitan

monooleate (Tween 80), cholic acid, acrylamide,

ammonium persulfate, bovine serum albumin, and

the bicinchonic acid protein determination reagents were

from Sigma Chemicals. Chelex-100 and TEMED were

from Bio-Rad Laboratories. Blue-Sepharose CL-6B

was from Amersham Pharmacia. Microtiter and gas-

tight syringes were from Hamilton. Screw-cap fluores-cence cuvettes and magnetic stir bars were from Starna

Cells. Septum caps were from Supelco. All reagents were

of the highest purity available.

Preparation of substrates. To remove trace metal ions,

all of the buffers and solutions, including 1M Tris ace-

tate, pH 8.1, and 10% (v/v) Tween 80, were made in

HPLC-grade water and further purified using Chelex-100

resin. Protoporphyrin IX and N-methylprotoporphyrinwere prepared as 200lM stock solutions in 0.1M Tris

acetate, pH 8.1, containing 0.5% (v/v) Tween 80. Ferrous

iron was prepared under anaerobic conditions as a 2mM

ferrous ammonium citrate stock by dissolving equimo-

lar amounts of ferrous ammonium sulfate and sodium

citrate [9]. All glassware used in the assays was soaked

in concentrated HCl overnight to remove all traces of

iron.Purification of recombinant murine ferrochelatase.

Murine ferrochelatase was isolated from an overpro-

ducing Escherichia coli strain transformed with a fer-

rochelatase expression plasmid containing a N-terminal

pentahistidine tag [27] and purified using Blue-Sepha-

rose affinity chromatography as described previously

[27,28]. Protein purity was assessed by sodium dodecyl

sulfate–polyacylamide gel electrophoresis, and proteinconcentration was determined using the bicinchoninic

acid assay with bovine serum albumin as standard.

Unit definition. One unit is defined as the activity re-

quired for the consumption of 1 nmol of substrate per

minute at 30 �C. Activities are reported as nanomole of

protoporphyrin consumed per minute.

Fluorimetric determination of ferrochelatase activity.

Ferrochelatase activity was determined using a Shima-dzu RF-5301PC fluorimeter equipped with a red-sensi-

tive photomultiplier tube. The thermostatically

controlled cell holder was maintained at 30 �C. A 2-ml

solution containing protoporphyrin IX in 0.1M Tris

acetate, pH 8.1, and 0.5% (v/v) Tween 80 was deaerated

in a fluorescence cuvette sealed with a septum cap and

containing a micromagnetic stirring bar. Deaeration

continued for at least 30min by applying repeated cyclesof vacuum and purging with ultra high-purity argon gas.

The cuvette was then placed in the fluorimeter cell

holder. Purified ferrochelatase was added to the mixture

by injection using a fixed-needle syringe, and the mix-

ture was equilibrated for 5min at 30 �C under constant

stirring. The reaction was initiated by injecting ferrous

iron, and the progress of the reaction was monitored

Z. Shi, G.C. Ferreira / Analytical Biochemistry 318 (2003) 18–24 19

fluorimetrically and continuously by recording theemitted light through an emission monochromator set

at 635 nm (5-nm slit width) upon excitation at 505 nm

(3-nm slit width). The rate of change in fluorescence

intensity (arbitrary units min�1) was converted to ve-

locity (nmol of protoporphyrin min�1) using a standard

curve of fluorescence intensity vs protoporphyrin con-

centration. The time course for each reaction was col-

lected using the Shimadzu software RF-5301PC, and theinitial rates were calculated from the linear portion of

the progress curves.

Steady-state kinetic characterization of murine fer-

rochelatase. The steady-state kinetic parameters KFe2þ

m ,

KPPIXm , and kcat of ferrochelatase were determined at

30 �C using the continuous fluorimetric assay as de-

scribed above. To determine the Michaelis constants,

KPPIXm and KFe2þ

m , and maximal velocity, Vmax the datawere analyzed in matrices of five protoporphyrin and

five Fe2þ concentrations by fitting the initial velocities to

Eq. (1):

v ¼ Vmax½PPIX�½Fe2þ�KFe2þ

i ½PPIX� þKPPIXm ½Fe2þ� þ KFe2þ

m ½PPIX� þ ½Fe2þ�½PPIX�;

ð1Þ

where KPPIXm and KFe2þ

m are the Michaelis constants for

protoporphyrin and Fe2þ, KFe2þi is the dissociation

constant for Fe2þ, v is the initial velocity, and Vmax is the

maximal velocity of the reaction. Eq. (1) describes the

steady-state velocity equation for a bireactant system in

the absence of products [29]. The kinetic constants and

standard deviations were calculated from regressionanalysis using the statistical program DATAFIT (Oak-

dale Engineering).

To calculate the apparent inhibition constant Kappi

of ferrochelatase for tight-binding inhibitor N-meth-

ylprotoporphyrin, initial rates measured in the presence

of various concentrations of inhibitor were fitted to

Eq. (2),

V0Vi

¼ 1þ ½I�Kapp

i

; ð2Þ

where V0 is the initial velocity in the absence of inhibitor,

Vi is the initial velocity observed at inhibitor concen-

tration [I], and Kappi is the apparent inhibition constant.

Eq. (2) is a simplified form of the Morrison�s quadraticequation for a tight-binding, reversible inhibitor when

the enzyme concentration is lower than the inhibitor

concentrations tested [30,31].

Results and discussion

The continuous assay described in this study for de-

termining ferrochelatase activity takes advantage of the

characteristic fluorescence properties of protoporphyrin

IX and the lack of fluorescence of the reaction product,

protoheme (Fig. 1A). Under the assay conditions andfor an excitation wavelength of 505 nm, the emission

maximum of protoporphyrin IX is at 635 nm (Fig. 1B).

Protoporphyrin absorbance at 505 nm is much lower

than in the Soret region, and thus inner filter effect and

consequent potential problems associated with unreli-

able interpretation of data for high fluorophore con-

centrations are minimized. In fact, fluorescence intensity

exhibited a linear dependence on protoporphyrin con-centration up to 12lM (data not shown). Collectively,

these observations led us to devise an assay for fer-

rochelatase using ferrous iron and protoporphyrin IX

and to monitor the progress of the reaction by following

the consumption of protoporphyrin as the decrease in

emission fluorescence at 635 nm with excitation at

505 nm (Fig. 1C). Iron substrate was maintained in the

reduced state by imposing strictly anaerobic conditionsduring the assay. In this way, the use of reducing agents

Fig. 1. Excitation (A) and emission (B) spectra of protoporphyrin IX

under the assay conditions. Protoporphyrin ð1lMÞ was prepared as

described under Materials and methods. The emission spectrum ex-

hibits a maximum at 635 nm ðkex ¼ 505nmÞ, while the excitation

spectrum displays four distinct bands at 406, 505, 541, and 576 nm

ðkem ¼ 635nmÞ. (C) Time course for the disappearance of protopor-

phyrin in the ferrochelatase-catalyzed reaction. Murine ferrochelatase

(176 nM) was preincubated with protoporphyrin IX ð2:7lMÞ at 30 �C,under strictly anaerobic conditions, as described under Materials and

methods. Reaction was initiated by adding Fe2þ (5:0lM). The de-

crease in fluorescence intensity at 635 nm ðkex ¼ 505nmÞ was contin-

uously monitored over the course of the reaction and the initial rate

was determined from the slope of the tangent to the initial, linear part

of the progress curve (see Materials and methods for details).

20 Z. Shi, G.C. Ferreira / Analytical Biochemistry 318 (2003) 18–24

was eliminated. (Thiol-containing compounds are com-monly used reductants, and they are known to promote

heme degradation [8]). Assays were conducted at near-

optimal pH [9,10,20], and the detergent, Tween 80 [0.5%

(v/v)], was included in the reaction medium to ensure

complete solubilization of protoporphyrin [6,8,12,13].

In the discontinuous ferrochelatase assays (e.g., spec-

trophotometric, fluorimetric, and radiometric proce-

dures), the rate of the reaction is calculated upondetermination of the product formed (or substrate con-

sumed) at the end of the reaction time. With this ap-

proach the progress of the reaction is ignored, and thus

for the calculation of a reaction rate in a discontinuous

assay it is assumed that the progress of the reaction is the

same during the assay time. We compared the ferroch-

elatase activity as determined, using Fe2þ and proto-

porphyrin as substrates under strictly anaerobicconditions, by the (1) continuous, fluorimetric assay and

(2) discontinuous methods. The discontinuous methods

involved (2.1) determination of the protoheme product

formed at the end of a 20-min reaction using the pyri-

dine–hemochromogen assay and (2.2) quantification of

the consumed porphyrin at the end of a 20min reaction,

using the fluorimetric determination of porphyrin (i.e.,

with the same settings for excitation and emissionwavelengths as in the continuous assay). After 20 min

continuous monitoring of porphyrin consumption us-

ing the fluorimetric assay, the reaction was terminated by

the addition of 0.5M NaOH and the amount of proto-

heme formed was determined using the pyridine–hemo-

chromogen assay. The amount of product (protoheme)

formed or substrate (porphyrin) consumed, as monitored

with either the continuous or the discontinuous method,was comparable. However, while the rates determined

using the discontinuous assays were comparable, the rate

calculated with the continuous assay was over one order

of magnitude greater (Table 1). These results confirmed

the importance of a continuous assay in the determina-

tion of rate of the ferrochelatase-catalyzed reaction.

Having established the reaction conditions, we set out

to determine the enzyme concentration range that couldbe used with this assay. The rate of porphyrin disap-

pearance was linearly dependent on the ferrochelatase

concentration over the range of 25–150 nM (Fig. 2). The

assay thus allows ferrochelatase to be used in concen-

trations approximately one to two orders of magnitude

lower than those routinely employed in nonfluorimetric

assays. Moreover, the calculated limit of reliable detec-

tion with the present assay is improved up to two orders

of magnitude over that of the pyridine–hemochromogen

assay [9,10]. An important aspect of any method for the

determination of ferrochelatase activity is to minimizethe presence of contaminating metal ions in the reaction

assay, since ferrochelatase can use, in addition to ferrous

iron, several other divalent metal ions as substrates

[12,13,20,22]. Clearly, this could cause an inaccurate

determination of initial rates at defined concentrations

of ferrous iron. Therefore, to remove potential trace

metals, all solutions and buffers used in the assay were

prepared in water previously treated with a chelating ionexchange resin (i.e., Chelex-100). Further, the reaction

was initiated with the addition of ferrous iron to a

preincubated mixture containing all the other compo-

nents of the assay. A stable reading of fluorescence in-

tensity before the addition of ferrous iron substrate

indicated that the subsequent decrease in fluorescence

intensity corresponded solely to the chelation of the

added ferrous iron.

Fig. 2. Dependence of the initial rate of protoporphyrin consumption

on ferrochelatase concentration. Initial rates were determined for re-

actions using 2lM protoporphyrin, 4lM Fe2þ, and various concen-

trations of murine ferrochelatase as described under Materials and

methods.

Table 1

Comparison of the continuous and discontinuous assays in the determination of ferrochelatase activitya

Continuous assay Discontinuous assays

Product formed (or substrate consumed)

(nmol)

35:2� 1:0

(heme formed)

40:4� 1:2 (porphyrin

consumed)

26:6� 1:8

(heme formed)

Ratea ðnmol min�1Þ 2:8� 0:1b 0:229� 0:006c 0:176� 0:005d

Assays were conducted at 30 �C using 176nM murine ferrochelatase, 5lM Fe2þ, and 2:7lM protoporphyrin.a Each value represents the mean and standard deviation of three independent measurements.b Initial rate as calculated from the slope of the linear portion of the progress curve.cRate as calculated from porphyrin consumption at the end of a 20-min reaction.dRate as calculated from heme formation [7,10] at the end of a 20-min reaction.

Z. Shi, G.C. Ferreira / Analytical Biochemistry 318 (2003) 18–24 21

Validation of the present assay was assessed by de-termining the steady-state kinetic parameters of purified,

recombinant, murine ferrochelatase. The dependence of

ferrochelatase activity on the protoporphyrin concen-

tration at various ferrous iron concentrations is illus-

trated in Fig. 3. From these data, the calculated Km

values for protoporphyrin IX and ferrous iron are

1:4� 0:2lM and 1:9� 0:3lM, respectively, while kcat is4:0� 0:3 min�1 (Table 2). It is recognized that the val-

ues for ferrochelatase activity reported in the literature

vary considerably [32]. These differences may result from

determinations with ferrochelatase isolated from differ-

ent species, use of different substrates, use of different

methods of preparing the porphyrin substrate solutions,

use of diverse reducing agents in maintaining the iron

substrate in the reduced Fe2þ state, and the reporting ofapparent vs ‘‘true’’ steady-state kinetic parameters.

However, despite the variability in the published kinetic

parameters for ferrochelatase, the kinetic constants de-

termined using the proposed assay were comparable to

the values reported for purified recombinant human

ferrochelatase [33,34] (Table 2). In contrast, the previous

kinetic analysis of recombinant murine ferrochelatase

using Fe2þ and deuteroporphyrin and the pyridine–he-mochromogen assay yielded Km values about 10-fold

greater than those obtained with the current assay ([28]

and Table 2). Probable reasons for the observed differ-

ences include (1) nature of the discontinuous assay,

which required a prolonged incubation (30min) [28] and

returned a lower rate than expected for a continuous

assay (Table 2); (2) the presence of dithiothreitol in the

reaction assay [28], which subsequently was shown toact as an iron chelator [10], and thus the effective Fe2þ

concentration in the assay reaction might have been

lower than that reported; and (3) the use of an alter-

native substrate, deuteroporphyrin, whose kinetic con-

stant is not the same as the physiological substrate,

protoporphyrin IX. Notably, similar differences were

also observed for the kinetic constants of bovine and rat

Fig. 3. Determination of the steady-state kinetic parameters of wild-

type mouse ferrochelatase. Assays were conducted at 30 �C using

100nM murine ferrochelatase and Fe2þ concentrations of 0.5 lM (N),

1lM (�), 2lM (d), 3 lM (O), and 4 lM (j) as described under Ma-

terials and methods. The hyperbolic curves represent the best fits to the

Michaelis–Menten equation of the data points regarding initial velocity

against protoporphyrin concentration while Fe2þ concentration is held

constant. The steady-state kinetic parameters of murine ferrochelatase

were determined to be KPPIXm ¼ 1:4� 0:2lM, KFe2þ

m ¼ 1:9� 0:3lM,

and Vmax ¼ 97:4� 7:9nmol min�1 mg�1 enzyme.

Table 2

Steady-state kinetic parameters for murine, rat, human, bovine, and yeast ferrochelatase

Protein/(source) Vmax

(nmolmg�1 min�1)

KPPIXm

(lM)

KFe2þm

(lM)

Porphyrin

substrate

Reducing agent Assay method Ref.

Murine FC

(recombinant)

97.4 1.4 1.9 Protoporphyrin None Continuous

fluorimetric assay

This

study

Murine FC

(recombinant)

136 95 112.5 Deuteroporphyrin DTT Hemochromogen

assay

[28]

Human FC

(recombinant)

165a 9.3a 9.0a Protoporphyrin Not specified in the text Porphyrin

absorbance

decrease

[34]

Human FC

(recombinant)

— 8.19a 9.35a Protoporphyrin b-Mercaptoethanol Hemochromogen

assay

[33]

Yeast FC

(mitochondrial

membrane

extracts)

— 5.9a 1.63a Protoporphyrin None; anaerobiosisb Porphyrin

absorbance

decrease

[13]

Bovine FC

(liver)

105a 80a 11a Protoporphyrin DTT Hemochromogen

assay

[35]

Bovine FC

(liver)

88a 54a 46a Protoporphyrin DTT Hemochromogen

assay

[20]

Rat FC (liver) 120a 28.5a 33.1a Protoporphyrin DTT Hemochromogen

assay

[9]

aApparent steady-state kinetic parameter values.bAnaerobiosis was achieved by enzymatic oxygen uptake [13].

22 Z. Shi, G.C. Ferreira / Analytical Biochemistry 318 (2003) 18–24

liver ferrochelatase when assayed in the presence ofdithiothreitol by the discontinuous hemochromogen

assay (Table 2).

Applicability of the continuous assay in ferrochela-

tase inhibition studies was tested with N-meth-

ylprotoporphyrin, a tight-binding inhibitor [35] and

proposed transition-state analog of ferrochelatase. The

determined apparent inhibition constant (Kappi ) was

94� 0:9nM (Fig. 4), a value that correlates well with anIC50 of 100 nM previously reported for purified murine

ferrochelatase [10,28] and a Ki of 75 nM measured by

zinc-chelatase activity in human lymphocyte extracts

[36]. The potency of inhibition for human and mouse

ferrochelatase byN-methylprotoporphyrin appears to be

one order of magnitude weaker than for bovine

ferrochelatase, which exhibits a much smaller Ki of

7 nM [35].Overall, the proposed assay offers several significant

advantages over existing procedures. Because the time

course can be monitored, this assay allows initial rates to

be determined from the slopes of progress curves, and

thus it eliminates the problem of nonlinearity in velocity

calculations resulting from prolonged incubation re-

quired for end-point assays. The use of an excitation

wavelength at 505 nm extends the upper limit of por-phyrin concentration permissible in the assay, as inner

filter effects are negligible. By imposing strictly anaerobic

conditions, the proposed assay permits the determina-

tion of ferrochelatase activity for the Fe2þ substrate

instead of metal ion substitutes, such as Zn2þ and Co2þ

[21–23]. The assay requires only a very small amount of

enzyme; thus, it allows economical use of purified en-

zyme stocks, and it also makes it possible to detect fer-rochelatase in biological samples of low specific activity.

In summary, the continuous fluorimetric assay pro-vides a sensitive method to measure ferrochelatase ac-

tivity using the physiological ferrous iron substrate. The

assay can also be employed in inhibition studies of fer-

rochelatase by metal ions, N-alkylated porphyrins, or

metalated porphyrins. Since the procedure can detect

low levels of ferrochelatase activity in biological sam-

ples, it should be useful for assessing ferrochelatase ac-

tivity in porphyria patient samples.

Acknowledgments

This work is supported by American Cancer Society

Grant RPG-96-051-04-TBE (to G.C.F). Z.S. is a re-

cipient of an American Heart Association Florida/Puerto Rico Affiliate Predoctoral Fellowship. We thank

Professor Michael Barber for helpful discussions.

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