A model has been proposed in which solid surfaces can act as a site for cataletic activity of condensation reactions for certain biomolecules. From this model, the adsorption characteristics of 5'ATP and 5'AMP onto the surface of CaSO4.2H2O was chosen for study. It has been proven that 5'ATP and 5'AMP do adsorb onto the surface of CaSO4. Studies were then made to determine the dependence of absorption versus time, concentration, ionic strength and pH. It was found that the adsorption of the nucleotides is highly pH dependent, primarily determined by the phosphate acid groups of the nucleic acid molecule. From this investigation, the data obtained is discussed in relation to the model for the prebiotic earth

Calderon, J.

Sweeney, M. A.

English

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An Investigation of the tiubulqj --:Lull vii raczer;L^^LC:^
FINAL TECHN
John Calderon and M. A. Sweeney A
Rh
1
a
a^
NCC 2-170
Dr. James Lawless
UNIVERSITY OF SANTA CLARAN
c/oSponsored Projects Office
Santa Clara, CA 95053
of 5 1 ATP and 5 1 AMP onto the Surface of CaSO4•2H2O
(NASA-CR-177005) AN YNVESTIGATION OF THE
	 N86-28994
ADSORPTION CHARACTERISTICS CF 5' ATP AND
5 0 AMP ONTC THE SURFACE OF CaSC4 Y 2H2O
Final Technical Report (Santa Clara Univ.)
	 Unclas
13 p	 CSC.L 07D G3/24 43301
1^
ORIOINAL	 -".^:!',
OF POOR'
`.
SUMMARY
A model has bern pp o posmd (LahAv and Ch8Ost 1982) in
which solid surfaces can act as n site for catalstic activity
of condonsati0n r, pctionq for certain bio0olecul g s ^ From
this node1v the adscr p tion characteristics of 5~ATP and 5/AMP
onto the surface of CuS ,^CQO was chosen for stud y , It has
houn p roven that 5~ATPand 5^AMP do adsorb onto the surface
o[ CcSO^, Studies were then made to determine the dePeO-
^jance of adsorption versus time, concentration: ionic
strenoth and PH * It was found that the adsor p tion of the
Uuclaotides is hishlu PH dep endeotn Prima p il y determined by
the phosPhato acid broV p s of the nucloic acid molecule, From
this	 investization, the dBte obtained is discussed in
relatior to the the model for the rrvbiotic earth,
.	 TVrVduation
/ The Possible rule of soluble walts in chemical evolution
~
i	 wos reoeOtlY d0cussed in relation to fluctuatin g prebiotio
' environments (Lah&V sDd Chant y 1982) ° In this g nVironmentv a
model was Pro p osed in which a h y dration-deh ydration system
could have been res p onsible for reactions of biomo1ecules°
Durin g
 the deh y dration sta g e of such a modelr some bio-
molecu1es tend to leave the solution and concentrate at
certain mtcromnvi yunments, such as micelles and a5wpegates'
at the Iimuid-was iOterface p and at the emer g in g
 solid
surfaces, In addition to inor g anic class y crystals of
sa1ub]e salts ma y have acted as sites foY hiomo1ecules to
leave the solution,
In a fluctuatin g environment, such as a tide p ool, lakev
or pond, some of the dissolved salts be g in to leave solution
and form solid cr y stals u p on deh y dration,
	
It is on these
-!
.^
|
^	
ORIGINAL
OF POOR QUALUY
.	
solid c py stalz that bimmolecuIms can adsorb and possibly
\\	 '` 	 rpact with one anothor to form orstar/ic comP1ex g s ° Then, upon
\
rc-htvdr^tioni, both the blomolecules and metal salts dissolve
k;-,ek into solution to PePeat the process,
In tine investi g ation (RishPon, O'Hara, Laha9r Lawless,
1923), it was f oVnd that 5 / ATP catal yzes the formation of
0l y oine u1i g owerr. ^ It was also found that addition of Msl C1~^
or ZnCl.	 increasud the Yield b y
 enhanciD^ the thormal
^
st 3hi1it y of 5^ ATP . It was thou g ht thot somehow the M~" arid
^+7o	 iuOS r g taPded the P ypol ysis of the of the ATP, which|
would allow a lroater concentration of ATP to react with the
^]ycinv^
i/	 Ti. was then a subject of curiosit y as to the means by
F
	
	
which t h p so]uhle saIt acted in retardin g this hreakdown°i
^
F,om the aforenentioned model, one,
 can conceive cf a Primor-^
d1^^ l8ke o p p ond cm0tain1VZ soluble ^alts, nucleio acids,
;4nd amino acids + DVPio g dehg drotion, the ooricentratioD of
the soluble salt soon reaches a point at which the salt is at
its saturation level. An y further deh y dration will cause the
6a] 1. to form its hydrated cr y stalsr on which the mdsopptioo
of biomolecu]es can take p lace, 5~A1P bein5 one of tham+
Once adsorbed onto the surface of the salt, condensation
rWactioUs can become more favorable due to the lower entropy
,	
.
of	 the	 system and enhanced thermal stabilit y of the
nu cleotide^
|
IL is with this scenario in mind that ex p eriments in a
	
.	 1a1.,1opatopy were conceived, Since it was thou ght that nucleic
| `^^	 acids and zomino acids were adsorbed onto the surface of the
-
Nje 3
^, exrmr^m^n6^ un p n devised ^m measure the a m ou nt-----	 ---'-	 - .^ -.---^.'- ..-^- -- --_- '_ -____.- '..- -.__..
.nd Qha y acte p istics of such adsor p tion, it aRy ^ The two
biomo1evW]es Noseo fo p
 this inYestifetiun We7e 5'ATP and
WAMP. Ori g inal studies of adsor p tion usin g
 the soluble salt
MdCl.
	
shownd uo measurable adsor p tion ^ It was then decided
W mse the leas soluble salt CaSO / in ho p es that a 1^ss
^
suluhle salt will p rovide a more sta"he surface apRa^
E^^p^rimeot^l
The nucleic acid 5'A T P was obtained
	
from	 KU.
Bioohvmica1s^ The VAMP was obtained from Boghpinger
Mannheim Pharmaceuticals, All salts were analvtical Quality
p easents and obtained from either Fisher* J ^
 To Baker y or
,
UvIliDckrodt, AlI other chemicals were of the PuTest commer-
vial 8uolit y
 available, All solutions were P p ePared With
filtered, ion-exchan g ed hQh p urit y water,
m	 ~Saturated solutions of CaSOy were p rePa p ed at 22 C by
.
^
	
oddin5 excess salt 0 8~24 1/100 mL> to the volume of water
	
^^
)desired, The solutions of lioVid Were allowed to eoUilibrate
oYeP[dzht to ensure saturation. All solutions of 5'ATP and
5 1 AMP were p rep a ped volumetricall y usios the saturated CaSO*
	
^	 '
'
as di1utent+
	
^
^
`
In all ex p eriments, 0 ^ 500 f ^ 001 g of solid CaSD; ~
	
_
2H^D	 i
was wei ghed into 10100mm screw-to p
 test tubes. To thisr S
five miI]ilitters of saturated CaS(L	 solution with the
desired concentration of the dissolved biomo1ecUIe ( 0 ^ 05 mM)
was added, If p H adjustment were necessaryv the y were made
^~^~	 at this time usin g
 1,0M (o p less when necessar y ) HC/ or NaOH
	
`
'. ,
^
`
^
	 PaZe 4
solutions. The test tubes were then sealed with a teflon
coated scrow~ca p and allowed to eouiIibrate^ The PH was|
^
wensVrod usin g a Beckman PM meter with combination elee-|i t^0de°
All exPerimmnta1 solutions were shaken for at least
w
throe hours at 22 C (room tem p erature) an @ standard hori-
|| wontal shaker, Ever y 30-40 minutes (exce p t for aamPle g that
were allowed to eQuilibrate overnidht) p each sam p le was vo p
-Wwud to ensure efficient miKins of solute b1oNo1g cuIe and
YQI!d oalt, and to minimize occlusion of the biomoIecule asi!
^ an im p urii ' v in the salt cr y stal lattice, After the aP p ro-
rriat g p nuilthretio/\ time' each som/`l g was c8ntrifused for
five minu t es on a st/Udard desk-U p ooeNtical cBntrifUle to
^	 ;eparate tho su pe,nataDt liaVid from the solid salt *
 Low!	 ^
apeoda were used as this affected the adsor p tion of the bio~
,
noWcUle ond the /H of the solution^
An Qiauot was Withdrawn and filtered throOsh 0 ^ 45 um
filters (Milli pore Cor p * ) to remove an y sus p ended salt
p art j c]es ^ The concen^P$tion of the sV pernatant lieUid s/.d
the ori g inal stock solution wa q measured b y ultraviolet
obsor p tionkmax = 259 nm for 5^ ATP @ p H 7 * 2) usine a Car y 14
double-beam	 peoordins sPeotro p hotomoter ^
	The amount of
|
	
	 biomuleoule adsorbed onto the solid salt was determined as
the difference in UV absor p tion between the initial stock
^
^ solution and the measured sam p le solution after shakins,
Ionic strendth mcasurement y
 were made usin g NaCl to vary
the concentration of inns over the solid salt. The salt NaCl
^
was added to the ori g inn^ saturated Cn^O|	 '	 ~, solution before the
'	 -
_Pose 5 /|\
-_'~`^	 -_.--^^--	 ^_	 ---..--	 -^	 -	 __"^-_ ..-~	 .rn^^u^ ^wu^^^urm^^uo ^u ^xuor^ ^nu^ vn^ ^n^w^^on was
urated ^ PH measurements were made of all sam p les after
su p ernaWRt lioVid was filtered and measured for absorb-
	
^ determining  ^^	 ^	 ^ the 	 _ ^'^^nce ^ ^n 	 t^e surface area of ^^e ^^lt^ ^ Q^2^^
0 solution or met ' ule0G blue indicator in saturated CaSO^^`
was used^ The absorbance of the meth y lene blue solution
662 nm) that was treated b y the same p rocedure as in
the p \'ecedinf p a p adraPh was com p ared to the stock methglene
blue solMon at the some p H value and the difference in
concentration determined,
Data and Discussion
In the first set of ex p eriments, 10,00 A of a 45 ^ 8 mM
^	 5^ATP soturatnd Ca^U` solution was added to 2 ^ 000 g of CaSO^"(	 -T
^
2H O. The final 5 / ATP concentration was measured to be 10.5
.	 ~
nM, which indicatod an adsor p tion of 176 nmoles WATP
adsorbed per tram of solid CaSO4~2H. O ^ It was this
ex perimont that first showed that the adsor p tion of 5~ATP
could be measured, It was then decided to do several series
of ox periMents to characterize this adsorption,
The next set of ex p eriments were to determine Nether or
not this measured adsor p tion followed a Lansmuzrian curve^
Ex p eriments were done to determine the adsor p tion of 5'ATP as
a function of final concentration. The results are shown in
fi g . 1 ^ An the g ra p h indicates, the adsorption is linear at
'
low concentrations~ and be g ins to level off as the concen-
tration over the solid salt increasos ° In fit, 2r a Plot of
^r
o kan g muir adsor p tion isotherm shows that the adsor p tion fo1~
A
xPr-,^:e 6
lows a Lz^n ,-I nojirian tidsot s vition y }.,,-tcept, at YeNj low concentra-
I.Ions4	 The bindin
g
 constant for the adsorrtion is Kb = 1*30
100r which indicates a very weak. interaction,
ADSCIRPT)ON j FINAL CONCENTRATION
VATP moto CaS4,?HO	 ORIQ31 NAL PA -GE  IS400 OFPOOR QUALITY
LANSOMUIR ADSORPTION ISOTHERM
AC
DO
W	 lu	 W^	 my	 ow	 CfC, 1^tw)
Fi ,-iure I	 Fissure 24
The ric-,;t interaction that wa;s loojhed at was the adsor p -
tion !a s z funt,, tion of ionic stren g th (fiEl f	 3> 4	 As extpectedy
ADSORPTION jq 10141C bTRENGTH
51 ATP eni. Cosq , 21110.
4
F
.cu
CV"
K,• I(y
k- 774 -16
T-2ec
m H.CI
Filure 3.
the adsor ption g oes down as the solubilit y of 5 ,'ATF, 
g
oes ur-
I wi
	
duo to the increase of ionic activit qj in the solution above
I
VAT  
	 ,wn
_
,
1 
om"'^
the solid salt#
\-L
	
	 The next p arameter looked at was the adsor p tion versus
t i0ie° Ph vorsUs tiiie was also measured from this eXPeriment^
/	 it wcs at this Point in the inwnstiZatioD that we decided to	 .
/
^	 ,-,1so ex, p er-.m g nt uith S"AMPr since this nucleotide was also
r-resent un the Pri0o pdiol ^arth°
	
The j , e6ults of the the
^	 Studg are shown in fi g , 4 and Fi g * 5^
ADS
.AusORPYIOm_ TIME
b'ATp_aS^^wp-^CASf4u1^O
~+-ImN".y3o(&..H
p^
5'xwp
a
^
^
"	 is	 xu	 26
""=°='
Fi g ure 4.	 Figure 5+
Usin g eouimola p concentrations of 5`6TP and 5^AMP, the
^ adsor p tion p lot shows a g reater (almost twice as much)
`
'	
adsorrtion for ATP than AMP^ This seems to indicate that the
adsor p tion is somehow 1inked to the eXtra two phosphate/
`
t,!,roups on the ATP molecule ^ Further, the eeui1ibration time
.	 for 5 1 ATP is much faster than that of 5'AMp ^ This was im p or-
 \
tant onl y to g ive information about how lon g sam p les should
^
JAW
k
0
	
P000 8
be enuilibrated fo p an the shaKe p when do0d further adsorp -
tion studiev,
From ris Sr one sees that eouilibration time for PH is
obout the same for ATP and AMP. The sha p e of the curves how-
• ver seem to follow the W p e of the adsor p tion curves * With
the thouaht that the adsor p tion is de p endent on the Phosphate
Krours and the match of the sha pe of the adsor ption and PH
Kurvos p it was thou ght that the adsor p tion is WON PH
Wendentt
This h ypothesis was tested in the next (and the most
difficult to control) sat of ex perimonts: the adsor p tion as
o function of PH* Due to the week bufferins ca p acit y ► the
dole Points between PH 4-6 were hard to stabilizer and as a
Penult y onl y had two hours of eQuilibration. The result of
thQ 0W adsor p tion vQrsQs PH are shown in fiz, 6.
ADSORPTION - pH
S'AlP
.41D.C#504 ZH'Q
PH
Fi5ure 6.
I ®rw-Uv	 NOV
IR
Pose 9
As uoe can weet the War p tion curve follows that of a
WW acid titration,	 The curve has an end point of -02P
which is suite different from the VATP PQ2 of 6,2 * The
dotted line shows what would have been ex pected if the
adsu p rtion followed the de p rotonation of the first Phosphate
Pruton,	 Howeyery this Qur ye does lend creedance to the
hvPothesis that the edwor ytion A linPod to the Phosphate
NFOUPS4
A contra;t
demon5trates an
the Phos phate Pr
pected ranne of
rat'; 2 a 6.3 (fit,
to VATPI; VAMP shows a CUPY0 that clearly
adsor p tion thA follows the dissociation of
Dtons, The curve Shows endPoints at the ex-
the PKg*s of 5 1 AMPP which are PKx1 = 2,3 and
7), From the PH of 1-2 y there is no
ADSORPTION w PH
pH
Fifure 7#
ranter the molecule is totall y Protonatedy
with a Positive char ge on the basic nitrowen of the supine
7 _7^
PnWQ 10
I
f
so
40
A 0j,
30
20
10
0.0
base, hround PH 2,3r the first deppotonation occurst which
results in the formation of a ZwittEr i on, This allows the
oxvien of the phosphate to form a we4v interaction with the
sell crystal#
From PH 3-6 y no further increase in odsor p tion is whowno
This	 is	 consistent	 with	 !he	 foot that no further
de p rolonstion is takin g, Placeo	 Frum PH W y a	 larde
odsor p tion is found, This is ProbaQ19 doe to the extraction
of the second ProWn which chan ges the VAMP molecule from a
Kitter ion to 4n ion with a neiative net chorket This
formation of the nevative char ge is im portent y for it not
an3v increavor the adsor p tions but it leads one to Postilate
that the on gAens on the Phos phate are somehow interacting
with the Co-	 ions in the salt lvttiQe,
In order to determine if the surface area did not chance
over the PH ran ge studied y MethAnne Blue indicatOr wes used
to measure the surface area adsor p tion verses PH (Fit, 85
ADSORPTION ,,, O.H
MMYLENLj3LUE_INDICAT.0R
t•
PH
Fi g ure Ot
it
K14,
p a ge il
As shownp the surface areo did not chan ge over the PH Ponze#
This hel ps to insure that the adsorption curves of both 51ATP
=4 I I AMP a p t not in error due to chonfina surface area,
Unc I us i on
At the data iodic:test G I ATP and VAMP do adsorb onto
the :urfacQ of 080-OH I G * The odsorp tion is hizhlw dep en-Y
Aunt on ionic stren g th, This would socoont for the lack of
wor5urobla adsor p tion onto MICI. - -tho Kish solQbilitw of
MaCl. would increase the ionic strunith to a Point that theti
nuclootide would bo Olmost totall y soluble * 	This is a
consideration that one must take into account when Postu-
311ind L;	 •01001. mechanism of adsorption in a CYCIO4
location*
The adsor p tion of the nucleotides	 is	 hiohlw	 PH
dwoundento This is another Paramet*r that must be taken into
vo Qount when loins covIins reactions, 	 The PH of	 the
h y dration watery the bufferins ca p acit y of the metal salts
useds dissolved Cl amount of water left in the dehydration
test tuber and other factors c;n Qhanse the PH of the
solution above the metal salt and thus chance adsor 3tivity,
The adsor p tion is Probable due to the interaction of the
ox y mcn- 3 hos phate moiet y interaction with the Ca V+ ions in the
crv5tal lattice, The treater adsor ption of ATP at enuimolar
concentrations and the PH de p endence of the adsorption
su pport this, While this interaction seems to be ionic ► the
low bindins constant (Kb = 1,3002) would indicate that the
bond formation is ver y weak  and thus not totall y ionic+
, I
0 <
low-
V *-):I a 1 00.1
Since it was the purpo%e of this investi gation to deter-
viine if 'ATP arid/or VAMP adsorbi zrid it sot under what
conditions y this line of ctF, erimontin g a^ duented concluded,
The direction t h ii t the e;., F-o j, i merits will now takL is to
QOritinuo w ih L, t- e cu o I i ri E; e;,'F c, r i men ts wero	 lost	 oxam i ned
( Pi P. h i, o n	 etk	 ftl	 Y and continue to e 4l ,.r-lore the cormitions
that _I ive tho boz t vield of e lwairie olizomorso	 Further
div ovcUoris vlsc, inc-Ifide usin g other nuclootidest metal saltsy
and tmir ►o vrids to find out what corabinat ions ,-,, ield the best
of	 biomoleculeso


An Investigation of the Adsorption Characteristics of 5'ATP and 5'AMP onto the Surface of Caso4 x 2H2O

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