Physico-Chemical studies of the activity of urease and the development of a conductimetric urea sensor

Jwmal~M0lecularLiquids.42(lBE9)83-98 Elsevier sdem

Publishers

B-V.. Amsterdam

2DepartmentofBiomedical Jiangsu, China-

83 -

Printed In The Nettherlanda

Souti-Kast

Erqineerj.ng,

Unb~ersity,

Nanjing,

SUMMARY

The hydration-dependence of the hydrolysis of urea by urease 3,5-l-5) has been studied using d.c, con&xtivity. a-c_ dielectric, hydration Isotherm, mass-spectrome tricandproton injection techniquea, Itisshawnthat~enEymicactivityof~e inneasesrapidlyforh~ationsgreaterthan 6wt-9 andthatthe supplyofprotonstotheenzyme istherate controlling factor- These results supporttheconceptpropasedby Carerithatthe critical hydration for the onset of activity of scme enzymes correspcrtds to the format3on of a network of proton percolation pathways on the enzyme molecule's surface, The hydrolysisof llreaby bIuuobilised ureasecanbenmnitored directlyasaresultofthe increaseddielectricpolarisability assocgated with the generation of electroactive zmm3nimnionswithin the bmobilisetdenzymestructure, Thisha8ledtothedevelopmentofa co~-~IuctImetric sensorcapableofdetecting 0.2niMureainsalt solutions of physiological ionic strength. (EC

Thisworkhasbeengreatly of Careri

et al

[l-3] cencernbq

transpertofprotons cur earlier

electrolysI8

~onictransportprocesses

theworkdescribeaheresuchmea hydration-dependence turn

the generation

inhydratedproteins,

solid-state

ofprotonicand

influencedbythetheories

has provided

and percolative

and it also

and dielectric Inprotein

follows

on from

rneasurexnents (4-61 structures-

In

surementsareusedtr-investigatethe

of the hydrolysis the basis

and studies

of urea by ureuL;e, and this

for the development

fn

of an a-c_

conductimetricureasensor, Urease

hyckolystzs

urea

accordingtothescheme:

(1)

Science Publiebera B-V. 0167-7822/89/$03.50 8 1989 Eleevier

side-reactionscanalsocccur,

_lY

El+

For aqueKYus solutions arethe

favouredprcxlucts

recognised

intheahoveside-reactions,

forsome~timethatdetectionofthe

carbonate sensors

ions offers

have

been

the basis

d escribad

Carsonate

when

Ithasbeen

alImlon3um and bi-

for a sensor

COXlfBlCtimetriC

of urea.

[7,83whichoperatebymonitorbx~the

~creaseinsoluti~conductivityaris~g ammonium

ions and CarlxJn dioxidazt

of pI%C8, ammotium

urease

fromtheproductionof fs added

to

Thesetypesofaensorareaffected'bythe presence fluids

of other

typically

ions have

in the test

solution

an ionic content

can severelylimittheir

practical

and,

equivalent

applications,

can also be seenthatprotcnsareconsumea, presence PH

of urea

to be determined

c1o-J. Here we report

of

function

studies

hydration,

dielectric,

mass vtric

account

than

that

studies

have

of urea

in physiological

develdped,

been

steady-state proton

fbnction,

to the development

detailed

-equation

this

(l)it

of urea

as a

by we,

cotiuctivity.

a-c,

injection

=Cwes-m increasedunderstandingofthephysico-

aspectsofenzym

relevance

to 150 mM NaCl,

byelectrochemicalmeammemenbof

and

fromtheirgeneralvaluetoan chemical

physiolqical

andthiaenablesthe

of the hydrolysis

using

since

used

of bfosensors_ given

We provide

previously

as the basis solutions,

independently,

suchmeasur~tsarealsoof

by Watson

[11,12]

here

for the conductimetric A similar et al

method

a more

of how these has

measurement also

been

1133,

MATERIALSANDMETHO~ Jack and 25% Vmalar*'

bean

urease

(EC 3,5_1,5),

bovine

(v/v) glutaral dehydewereobbined grade

urea

serum

albumin

fromsigma

andsodiumchloridewereobtainec?

(fraction

chms, from33DHLtd.

V)

and

For the solid-state

electrolysis

ureasewasdialysedthree

times

lyophilised

and

compressed

l-33

m2_

Thetsedisc

x

10-4

loaded

spring

against

distilled

studies

waker

at l-3 x 108 Pa into discs

electrodes

electricalluea

and dielectric

at neutral

of surface

in ateaperuture-co

r&rolled

described

intoavacuumand

fully elsewhere

_

[5,14],

fregllency

range

10-4 Hz to 60lcHzus~gtechnig[uasalsod~ribed~~ere_

[X5],

and

hotherms

were obtained

using

a cahn

electrob&ance

installed

inavacuumsystemwhoseovarall

was controlled

to within

O-1 K and where an ac cmratecontrolof

partfalpressureofwatercouldbemaintained, obtained

for ureaset,urea,and

temperature

Isothermswere

formixturesof

ureaseandurea,

mb&ureswerepreparedbythemechanicalmixiugof urease

urea,

and

contab-bem*at

area

and vacuum-tight

incorporated

Mea~~ofthediel~fcp~rtias~~remrtdeinthe hydration

PI-X,

sampleswerethenretainedbetweentwo

surementcell,whkhwas

mass-spElctroma ter system

the

followed

These

p =-%MW

by freeze drying

and storage

in vacuum

t&ght

4OC.

The conduct3_nu3tric ureasensorwasconstrucbdbyimuobilis~g u.reaseontoan adding

wt.8 bovine

Thiswasachieveclby

zurayofgoldticro-elmes_

150 IDM N&Cl

solution

to a mkture

of 25 wtm9

serumalhuu&n,untilathickgelwas

wasusedto

apply

this

gel,

as

formed,

earxay

as those

used

qlutaraldehyde HIM NaCl

150

tilution

solution

to

Othermethods

-98,

then immersed

for 30 seconds

E3amadesign studies a

into

[16],

25%

(v/v)

ardthellrinsedtkloroughlyh

ex#sfzgl-aehyded-

we for

0,2mm,

fabricatedonsttandard

iu our dielectrophoresis es were

Theg%l--covaredelectmd

around

wereofthe

printedcircuithoard.Themicro-electmdes

75

plus

Anylonbrush

a thin film of thiclamss

ontoanintePdigitatedmicm-elecbcd and dimensions

urease

fmmob5lis5ngureaseontothemicro-

electrodaswere~iga~,~cludlragthaBtandardmethodd~ibed by

Broun

the

[17],

but

mostrobustand

the

procehre

outlined

atmve

films,

monitored

on beLug

using

as well as with Ltd)

specifically

to provide The

reprcducibleresultforourpurpose~ of

~theele~~~lcorrductancaandcapacitance -se

appear&

a

iuser-ted into solutions

frequency

response

analyser

an kqedance

analyser

design&

to interface

these

changes

Milked

containing

urea,

were

(Solartron,

model

1250)

(node1 108, Orbit

Biotechnology

with conductimetric

sensors-

86

Physico-chemical

studhs

hydration

The

for

isoth-

analys%d

mixtures

tare

obtained

forthemonolayerhydration

Values

according

of the monolayer

25oC hydration

and

to

B,E_T_

parameter

is

20% urea

4-02

30% urea

3-87

bean

urease

with

one active-site

[19]- Based

value

(5-98 wt%

binding

H20)

of 321water

shown

3n

has

enzymic

lx,,, Cl83

a molecular

wetight of around

nickel

Ion per

weight

of 96,600,

obtained

for purified

ureasa

conmspo

The

per ux-ease slllnmit,

weight

The

gain,

reaction

been measured

of umase

and this

urease

Negligible

enzymic

-tothe

Typical

isotherms +

occurred.

Thahydrolysisofthe fnoppositiontothe

effect

of the urease rates

by Skujins

lyophilimd

h,

waalimitedtothehydrationlevel

activity

hydrati&-dependence

the

extenttowhichtheisothermsfortheurease

ureaby~~~resultedin~leweightl.o9s hydration

590,000

cam 96,600-daltmn

molecular

ureanliJ&urescouldbed~ at which

in the

on a subunit molecules

Fig-l,

hm are given

values

5-58 4-88

Jack

[la] amI

(w-t,9 wutelr)

u.r%as%

ahemamer

subunit

are

hm

1010 urcm

Native and

theory

for ureaseandurease+urea

Sample

pyre

the

parameter

hydration

isotherms

various ur%a/ur%aset

ur%ase

and

of urease

was used

activity

shown

activity

was

to various ob6erved

by

have

eqmsed

humidities them

the

in Fig.

atlowhydxations

and McY_az-en [20], who 14 C-urea

to derive

b&w

2, pmzviously

mixtwxs at 20°C60%

YZhtiVe

of

87

24

22

20

tbI1

1

a

Pig-

I

0

10

Hydration

1

and

humidity, occur g-=-Pat 60% water

20

isotherms

(c) urease

and

it

the

relative costent

40 daKvc

50 humidity

obtains

was concltied hydration humidity

In a urease

that

exceeding

70

hydrolysb

90

(a} urea-,

of urea

for urea~e concluded

+ 10% urea

80

(b) urea

mixture.

ona molecule

is&henns it can 5

60

at 298 K for

+ 10 wt,% urea

forwatercontents From

30 ‘!A

e

could

Per urease and urea

that

almost

only

polar (Fig-l) then

all of the

isboundtotheureaae

8B

it is evident

m>lecule~Therefore, activity arouml

of urease

from Tzibb

that

the

to a BET monolayer to loot hydmtion

1 v

Thereactlonratecorraspanaing

in Ffg_2 was calculated quoted

(Fig-a)

inmxawrapidlyafmitsh~tion~

6 w-t,%, which

anmrageofwuter~

2+C

from cmr work

Iq sigma

f'rcm the

full solutbn

act5v5ty

at pH 7 and

(r-a. 1pMunitlibarates1@5ofNH3fromurea

perminute). saiuplasof

urease

+ 101 urea

-

hydratd

between

toaMterconterrtaenanr~11~,las~sible, weight C&n

1~

a~~~iated

RI ectrobalanca~

and a value rmmlt

close

rqortecl

Fig.2

of 44.3+5

-xi=

Tl-dsprovicledthe

was mcmibced

activationflot

)cJ/mole for the activation

to thuu3e 02 33-5 kZ/mole

[21] ard 46.0+5

by urease

for experimental

using shuwn

energy,

32OC

details,

the

in Fig-3

which

m-la

in solution

'Hydrationdepend~ofthehydrol~isofureabyurea~at 298 K, See text

ad

andtherateof

urz% hydrolysis

forthehydrolysisofurea

19%

is a [223

conditions,

The

of Figs.2

results

hydrations also

&3

far the

are of use, not only

activity

of urease

for the development

at low

of biosenso:_'s, but

for eurtherurade~~dingofthaphysico-chemicalpro~s~that -

controlenzymea~ivity.Oneparti~arly important Careri

biophysical

et al

activity

[33

concept

interestingandp&ntially

related

that tha critiad

corresponds

to Fig-2

hydration

to the formation

is the proposal

for the onset of enzyme

of a network

ofadsorbedwatermoleculesonthe

by

of percolation

surfaceoftheenzyme,

pathways There is goodevidenoethatsdao~wat~TiZ01~P4caJlpravide

I

3.28

I

330

I

3.32

334

1000 /

Fig-3

I

I

Temperature

dependence

a hydration

content

T

I

3_36

I

3.38

3.40

1

3-42

I K-l)

of the hydrolysis

of 11 wt.%

of urea by urease

(dry weight

basis),

at

g

Hz0 / 1OOp ureut + urea

I 3.4

I 5.0

I 6.4

I 0.7

0.3

0.1

0 0

Fig-4

10

AInountsof mixture (H20 us&

ammonia

of urease

20

30 % relaHve huml&ty

and carbon + 10 wt.%

as reference

from

released

at

293

as a function

of

hydration.

dioxide

urea

40

in mass-spactrometry)

K

a

91

of

scheme

(I), l3w rmlef3 of ammonia

-ation

one of carbon

dioxide,

and 30 these

mixture elUpliC

that

that

ammonium

Studies that

Evidence

detected

resull23 indicate

for each

.urease+~ealnixture,

ions are retabmdwithinthe Electrical

can be prcduced

by monitoring is shown

of amuuJn~umionscanbe

th~enzyraicgeneration the

electrical

conductivity

of a urease+urea

In I?ig_!S-At, and above, the hydration

aclxivity occurs

the d-c. conductivibg

level

of the mixture

at which exceeds

of puJreurease,

-12

Wt. Fig.5

HydCE&tiOn-depend~~

(a) urease,

Anexperimentwas the

urea

just detectable,

H,O

of thy d-c. conductivity

and

(b) uremse

+ 10 wt,l urea

alsoperformedto

activityofureasedepends sample-

%

using

mixture,

fnvestigatetowhatextent

onthesupplyofprotons_

hydratedtoalevelatwhichthe and

at 298 K for

a proton

injecting

Aureasekl08 enzymic

activitywas

electrad e [I43 protons

were

into

injected

conductivity

of

magnitude, is go&d

active

the

the

-

sample,

sample

Ucrmmed

Thiseffectdidnot Efvidence

sites,

1191

As by

occur

for

Each

ahawn

at

for

samples

(1)

rapid

pathways

Furthemore,

-6

for

factor shown

percolation

indIcatesthat

in

theory

the

percolation

Figure

of

1

et

.

s-”

protons

scheme

6 provides

Careri

m-.e--

suggests

to

the

supplyofprotons

for the reaction of

6,whichcan

uxxaselknoleculeprwide

al

for

the

active

sites.

istherate

of equation

support

(l),

As

such,

proton

[3]_

m~m-m-

_-*--cc----

l

”

YBiere

at leastsix

increaseoPenzymkactivitp,

itwouldappmkrthatthe

controlling

of

umzafze-

posfxmsing

urfSasemlecule

thnt~hewut~~l~esadsorbedonthe

-b

pure

increaseofumductivityshcmminFigum

beassurmdtoreflecta

effect

of

the

omIen5

andprcrtonsmustbe~dilynvailableatthesesites,

TI-mstep+;Lse

the

Fig-6

five

andth~rea~onscbemeofequation

watermoleoules

efficient

in

least

l

,-rn 5

* -l2

t -15

Fig.6

t

The

change

sample

in

d-c-

inducf&by

datailrp,

conductivity

proton

of

injection-

a urease

See

tmct

-I-10 wt,%

urea

for4zxperimental

Thecbminantfea~oMainea meammmntsareshowninFigures7

70 Q

60

d E

.. 50

C

CO

/\

A

a

]]

30

\ \

20

8 8 8

\ \ 8 .

10

.

\

\

\ \

0

Fig-7

I

I

-3

-2

1

I

-1

DieCkct~ic (c) 12-4,

loss peak

1

for urease

2

inthese

figuresappears

b

3

at 298 K-

(a) 13.6 arta (e) 18.3 wt.%

Thelosspeakf3hown

1

I

0

(a) 8.5,

5

(b) 10-4,

hyckation,

to&the

So-calleJd Q-

dlspersi.onthathasbeencharactariseaforp~~rmche~8bavina plasmadLburmLnandl~ozyme,wfioseorigi.nhasbeenconcl~~tobe associate&I with results

for Fig-7

relaxation feature

frw, urease

are

frequencywith mixture

the loss peak activity_

transport

slowly

The

effect

where

awell

appearing

at a hydration

Clecreasec¶ in both

almost

of protons

[2-61, The

ureaseandtheincreasekofthe

Tncreasinghydrationis

(Ffgs8),

is therefore

hopping

for pure

of the a-clkpersfon~

urease+urea water

the activated

certainly

near

characterised 10 kHz

of around

magnitude

relatecl to the

and

for a

12 wt-+ relaxation

onset

of

94 60

-

Uf** co

-

30

-

20

-

lo-

OL

I

I

-3

Fig-

8

-2

-1

0

DiellectriclosspEIak (a) 5-0,

(b) 6-4,

57.6 r-h_

Closer

study

for

of these

loss-peakincrea investigated

I 2 Lop, c Cltzl

for urease

(c) 8-7 wtl hydration,

(d) 9 hrs,

temporal

and

(e) 66 hrs.

effects

revealed

features

further,

beconsktentwithreactionschenm whichareboth

frequency

of the a-dispersion

NaCl

solutions

Urea

factorslmownto

imnobilised

ureas43

containing

frequency

5

urea

at 298 K-

Sample

hydrated

that

initially

at

the

Wehavenot

at first

sight

they

appear

to

apH

influencethemagnitudeand

in lysozyme

[3,6],

Sensor

elelctrical impedances Solartron

but

4

(1) involvingdehydrationand

increase,

various

+ 20 wt,+

sedinmagnitudebeforedecreasing,

these

Conductlmetr~c

3

were

different

concentrations

monitored

response

conductivitywereobtainedfnthe

electrmleswereimmersedin150mM

analyser,

from

and the&z

10m2 Hz to 50 kHz usfng

Tim greatest

frequency

of urea.

range

changes fzomlto10

a

in k.Hz,

96

which

is

consistent

with

the

are

shown

Corductivitymeasurements, operathg

at

1

concentrations

kHz, In

temporal

effect

shown

in Figure

0btainedusinganOrbit

150

mM NaCl

in Figure at

8.

ImpedanceAnalyser

9 for

a range

of qrea

18OC,

15 100 mM. wca

110 VI E g

105 -

t -z s 100 .

0

10

5

time

Fig-9

Change on

with of

immersion

ccxductance into

of urea,

temperature

conaitions

In salt

conductivity

solutions were urease

record~I

immob5lised

was

Insertedintotheureaaolutions~ 9czmresponded

of

immobilised

the

the

surrounding

of

inmobflised solutions

the

s ensor

of physiolqical

the

Figure

Imins)

I_50 I#¶ NaCl

comzentrat~ons

stable

I@I urea

inllmz

placed

when

a gold

urease

electrm3e

containkq

could

ionic

different

readily

detect

strength.

mi cro-electrode

No

O-2

changes

system,

with

aroundbutnotccrveringtheelectrcdes, Thisindicatedthatthedataof

toelectricalchanges urease

l5

film,

solutionwasnotbeing

and

occurringwith~thebulk

that

a conductivity

recorded_

increase

of

Acammonmethodofentaluating~zymeacUvity~~~i<3B~by application

of the Michaelis-Menton

Such

is given

a plot

in Figure

ofsubstrateconcentration reaction

rate,

1

-lb

Woolf

plot

[S) intercept

whose

value

the maximal which

is close

rate.

to the

range

urease

-from conventional

higher

Michaelis

immobilised surfactant

anduxease

membrane

than that

most probably

result

from

20

5s

[S] fs the Urea

the Michael%=

(Q-5mM) obtained

for G

constant

G

yielding

half

is 6-9 mP¶, for Jack

bean

1243. A slightly

[25] for urease

comparedwith inahydr

[26] an apparent

a reduction

tion

reported

studies

fzoluble enzyme,

thematrixsurroundIngtheenzyme~

where

the value

kinetics

immobilised

exhibited for the

Fig.10

gel,

the

I

I

gives

of 5mM was

in polyacrylamide

solubleenzyme, greater

constant

is

rateofchangeofconductance-

of values

enzyme

v

in Fig-lo-

substrateconcentra

From

9. A plot

where

1233,

urea)

plot

tothe

of Figure

15

of Fig-lo, the

plot

line,Zl.SiSthEt-

imM

is

the Woolf [S]/V,

I

in the Woolf

reaction

ratio

lb

of the data

co-

usbg

the data

the

!

S Csl

concentrationandv

The

versus

a straight

give

0

Fig-10

10 using

[S]

should

theory

4 mMobtained

forthe

cxarbon-based

liquid-

I$,,value

50 times

These

increa sed~ValUes of the diffusibility of urea

Thus,

the results

of Figs-9

t 10

in

show

that

the

urease

reflected

our

immobilisation

sample, the

procedure

did

and that the bpedance

hydroly&

not deleteriously

affect

measur eraent accurately

of urea by ureas&,

C0NcmsIoNs The

hydrolysis

of urea by urease

chamicala~physicaltechniquas, rapidly

as the hydration

(Fig-2)

and

wt.%

the

enzymic

corresponds

conditions_

observed

on proton

activity

water active

The that,

hydroly&_s Also,

the

molecules

ammonium

of urease

increas&s

of the enzyme

is raised

above

energy

(Fig.6).

ustig

situation

irmnobilisation

ammoniumions

the urease results

to electrically

the of protons

form

shown

structure.

in Figures

5 & 8,

the generation

the basis

of

for a sensitive

in J_50 mM NaCl

solutions.

Our blood

testingtheseusorohhuman contentsarearound5mM

and ihvefzMgal&q

is relatedtosuch

(Fig-l) suggest molecular

monitor

O-2 mM urea

how

factors

the sehsor

sensitivity

as electr&fedesign

procedures,

Thisworkhasbeensupportedin

part

by

the

Basic

Foundation

for Cancer

thank

Ur S-Bone,

Mr T-A-K.Wray

for valuable

of

generatedduringthe

Programrae of the National Professor

where

for the percolation

plasmaandurinesamples,whosenonnalurea

enzyme

for the proposal

for the onset

a mass-spectrometer

presenteffortsaredirectedtowards

and reproducibility

effect

molecule,

and dielectric

and 350 nau~respectively,

of '11

is in full

the dramatic

support

hydration

are r-etained within

of detecting

urease

provide

6 wt.%

at a hydration

with

correspo nds to the

ions and that this can

capable

when

together

form pathways

it is possible

(Fig,3)

observed

pH conditfons,

conductivity

such

The activity

results,

of the enzyme

ubtati

of urea

that

sensor

enzymes

normal

show

to that

These

injection

sites

results

under

by several

[3] that the critical

of many

adsorbed to the

et al

monitored

activation

closely

solution by Careri

content

has been

G_Careri,

discussions

and advLce_

science

Research

(USA). We

and Miss

K-Adams

and

98

G_ Careri, P_ Yang and J-ARupley, Nature, 284 (1980) E_ Gratton, 572-573, and J-A_ Rupley, 2 G_ Careri, M; Getraci. A_ Gi ansanti Acad, Sci, USA 82 (1985) 5342-5346, I ?roc_ Natl, and J-A_ Ru~ley, Proc, Natl_ Acad, fici_ USA 3 c:_ Careri, A. Gimmti 6810-6814, I 53 (1986) r_ Bahi, S. Bone, H_ Morgan, and R.Pethig, Int_ J_ Quantum Chem: 4 &mnt_ Biol_ Symp_ 9 (1982) 367-374, 3, MorganandR. Pethig,J_ Chem, Sot. Faraday-. I, 82 (1986) 5 143-156, r_J. Hawks and R_ Pethig, Bicchim. Biomys_ Acta_ 952 (1988) 6 27-36, Chem 33 (1961) 1757-1760, 7 #_ chin and w. KbToont-je, Anal_ J. Cherm, Phys_ 65 (1965) 673P. Bourelly anB v_ Bourelly-rxlrana, 8 677. 9 Z_G_ Guilbault and J.G_ Montalvo, J_ Am, Chem, Sot, 92 (1970) 2533-2538, 10 J_P_ Joseph, Mikrochimica Acta 11 (1984) 473-479. 11 J-J_ Hawkes, Z-H, Iu and R_ Pethig, Abfztr. BIO'86 Sira. Conf_: obxluxolcqy, mndon, 1 Probes for Detection and Measurem entinBi (1986) 25-27, R. Pethig, J_A.R. Price ti T_A_K_ Wray, in: J-L_ l2 B.A. Iawton, Electrostatic Charge Migration, I_O_P_ Series, 14 Sw=bn (m-1, (1988) 21-31, 13 L-D_ Watson, P_ Maynard, D_C_ Cullen, R.S_ Sethi, J. Brett&z and C-R_ Zcrwe, Biosensors, 3 (1988) lOl--115, In&rum_ 19 (1986) 8014 H. Morgan and R_ pethig, J_ Phys_ E_: Sci. 82_ 15 P. Carnachan and R_ Pethig, J_ Chem, Sot_, Faraday Trans_ I, 72 (1976) 2355-2363 16 J-A-R. Price, J. Burt, and R_ pethig, Biochbr~ tiophys_ Acta_ 964 (1988) 221-230, Methods En~ymol_ 44 (1976) 263-280, 17 G-B. Broun, 18 S* Brunauer, P-H_ St and E. Teller, J_ Am, Chem_ Sot, 60 (1938) 309-319, 19 H.E. Dixon, 3-A_ Hinds, A-K_ Fihelly, C_ Gazzola, D-J_ Winzor, R.L. Blakely and B_ Zemer, Can_ J_ B&chew, 58 (1980) 1323-1334, 20 J-J_ Sku-jins and A_D_ McLaren, Science 158 (1967) 1569-1570. 21 R-J_ Miller, C_ Pinkham, A_R_ Overman and S-W_ MO&, BiochbBfophys_ Acta 167 (1968) 607-609, 22 K-R_ Lynn and P-E_ Yankwich, Biochixn_ Biophys_ Acta 56 (1962) 5l2530, 23 F-B_ mtrong, Bfoche~&~try (2nd_ Ed) Cxford Univ_ a, (1983) 136-139, 24 F-J_ Reithel, in: P-D_ Bayer (Ed.), The Enzymes, Academic Press, New York, 4 (1971) ?-21. 25 G-G. Guilbault and J. Das, Anal_ Bia&hem_ 33 (1970) 341-355_ 26 S-W47 (1972) May and N-N_ Li, Biochem. Biophys, w_ Commun_ 1179-1180. 1