Ultrasonic relanation studies in aqueous amino acids

4.1. INTRODUCTION:

Amino acids are cornmon conlponenis of all orgairi$rm~s. I+mtein oi ail byecies,

from bacteria to humans, is made from the same set of 20 stitniinrd amino acids.

i2roteins play many different biological roles in [;\ring s!.stern5. They are the molecular

instruments through whch the genetic information is expressed. Ail proteins are

constructed from the basic set of 20 amino acids, covalently linked in ~h~~racteristic

sequences. Because each of these amino acids has a distincti1.e side chain ~vhich , fends

it chemical individuality. The group of 20 amino acids .i.vfiich form the basic building

blocks of proteins may be regarded as the alphabets of protein structure. The most

remarkabie thing is that the cells can join these 20 amino acids in many different

combinations and sequences, yielding the peptides and proteins with strikingly

different properties and activities. Amino acids in aqueous solution are icnized and can

act as acids or bases. Knowledge of the acid - base properties of amino acids is

extremely important in understanding many properties of proteins [l]. Besides, the

biological systems consist 70% of water; the study of interaction of these amino acids in

water has become prime important.

Ultrasonics has become an important and widely accepted method for non-

invasive imaging of human body and thus offers greater potential for further

development in diagnostic medicine. Ultrasonic energy has the ability to propagate

through soft biological tissues suffering only moderate attenuation in its passage. For

this reason, it has been widely used for measurement and imaging in the medical

context. The propagation of an acoustical signal in biological media is characterized by

variation in physical parameters which describe the state of the tissues. The ultrasonic

propagation properties of the tissues are governed to some extent, by their biochemical

composition [2]. Thus characterization of the materials by the determination of

ultrasonic wave propagation parameters is encouraged.

Lniierstar~ding the stabllitv oi ihc native sirucitjre 0 1 prtrtcii~s a:ld iur~f~rn-isiion

of (??her biopolyrners in aipeocis sol~itioa~ are of i u n d a n w ~ ~ t , ~ i ~n~porta:>ce A~l'jny

bioivg~cal phenomena have been ii?e subject ot great ci~,lilengc ;lnd extensive

in\.est~gatio~~s 13-61. The state of- ionization of the surfate ot bro rnoiecules and reiated

hydration and proton transfer or these sites are sigiuticant parts of ihe interactions of

these rnoiecules with their environment. Research work on ultrason~c studies 17-?5J,

partial molar volume studies jl6-IS] and heat capacnty studies 119-211 of aqueous

soiut~ons ot amino acids have been carried out to understand their heha\.ior in an

aqueous env~ronment.

Ultrasonic techniques have already been ernployed successfully to observe the

dynamic equilibrium between multiple ~someric forms of moiecules and to arnve at

more complete kinetic descriptions of chemical and structural reactions whose

relaxation times are comparable to the period of the ultrasonic wave 1221. The adiabatic

propagation of a longitudinal acoustic wave through a fluid medium result in time -

dependent, localized changes in pressure, density and temperature. Thus, the brave

motion may perturb molecular equilibrium at rates which depend upon the sound

frequency. For a non-ideal fluid, this lead to a time lag between an applied pressure

and the ensuing changes in the density. Consequently, molecular energy level

populations are perturbed at the expense of acoustic wave energy, and the process is

referred to as absorption.

Ultrasonic absorption spectroscopy has long played a leading role in research

involving the kinetics and thermodynamics of proton transfer reactions since the

relaxation times of these processes lie in the region of time covered by this experimental

technique.

Only a few biologically important macromolecules have been subjected to

thorough examination. The acoustical investigation of Casrtensen et a1 (231 on aqueous

hemoglobin solution indicates that the interaction between the solvent and the solute is

the prjnc~pal mechanism ot acorrst:~ i ~ l ? s ~ r p t ~ ~ i i ttl ii.~t>se s;,.h,terns., the

pp*btrre vanation assoc!ntrd !tV!th !he sound iv'3t.e perliirbi the eqi~~libr~inm

Jlstnbutlon of soi1,ent n-ioiecuies that are izeakly bonded to the solute, ,~r-id since

rearrangement of the solvent molecules does not occur ~rrsi;antane~usiy, absorption

results A slrniiar situation of excess uttrasonlc absorpt~on due to the interaction

bet~veen the solute and solvent rnolecuies is reported in aqueous detrar l soiutlons by

Hatviey et al 1241

'I'he acoustical studies carrled oiit by Kessier and Dtlrara 1251 on aqueous bo\.lne

serum albumin (HSA) as a function of frequency ha1.e sho1z.n that there is a sharp

increase In the excess absorption as the pH of the aqueous BSA solutlor~s are reduced

below pH 4.3. A similar increase in excess absorpt~on 1s also observed when the pH of

aqueous BAS is raised above pH 10.5. The increase in excess absorption as the pH of

aqueous KSA solutions are reduced below pH 4.3 and increased above pH 10.5 is found

to be reversible in nature in this range. This effect is found to be more pronounced at

low frequencies. The increase in absorption below pH 4.3 is attributed to the

intermediate N-F' transition and the increase in excess absorption above pH 10.5 are

described as due to the expansion of BSA molecule.

Many workers [26, 271 carried out ultrasonic absorption studies in aqueous

solutions of proteins as a function of frequency and at different pH ranges. These

studies suggest that there is an excess absorption of ultrasonic waves at extreme pH's of

aqueous protein solutions. These workers attribute this observed excess acoustic

absorption at extreme pHs of aqueous protein solutions to the perturbation of inter-

molecular proton transfer at ionizable side chain groups. However the mechanism of

acoustic absorption at neutral pH has not been accounted for quantitatively, with

solvent structure mechanisms, proton exchange and Internal motions of side chains

being proposed (281.

Hussey and Edl:londs i29j irorrl their altra~nraic ~nre>i!? ;a t~oi~ ui p:otc)n tr,ir~ster

reactions in aqueous glycine sol~rtiilns !n t1 .1~ pH r,!i-i&e b N :n 7 7 !7,si t. ~ c ? i i u i ~ ; J t ~ ~ l !ha: the

proton transfer reactions at amino and carbmt.P g ~ o u p s i ~ ~ r ~ n o t be responb~bie for

signit~cant contribution to the total illtrason~c absorption 'i'his range ot pH is

considered as the physiologicai range of pH values of blood ?hat 1s cornpi~!itile witla life.

A similar result of negligible contribution to the total ~litrasonic '3hsorption drie to the

proton transfer reactions has been observed bv the sarile authors 1261 in aqueous

soliltions of proteins in the pH range 6 - 8. This inl.estigstion on ailueou5 so!utions of

protein and polypeptides also establish that the proton transfer processes are very likeiy

a major mechanism contributing to the excess absorption in the range pH<6 and pH>&

However, in each case it is likely that this is not only the mechanism In effect, but other

rnechan~sms such as solvation equilibria, conformational changes and keto - en01

equilibria may also have their contributions superimposed on those due to proton

transfer reaction. But even then proton transfer cannot be eliminated from

consideration.

The theoretical and experimental studies of Holmes et a1 1301 on cysteine at

neutral pH indicate that in contrary to the studies of Hussey and Edmonds [26, 291,

there is an excess absorption in the neutral pH range 6.5 - 8.5. This excess absorption at

neutral pH range of aqueous cysteine solutions has been explained as not due to the

proton transfer between the ionisable groups and the solvent hydroxyl ions, which will

not produce significant absorption. But it is explained as due to the intramolecular

proton transfer. This intramolecular proton transfer may be significant at neutral pH

where ionisable side - chain groups are present. Similar ultrasonic absorption studies

are carried out by the same authors [31] on aqueous glycyltyrosine solutions in the

frequency range 2 - 50 MHz and in physiological pH range (6.8 - 8.8) and at a

temperature of 37°C. The excess acoustic absorption observed in glycyltyrosine

solutions in the physiological pH range is attributed to both inter- and intra- molecular

pfi:!on transfer processes ~!v.ulving U. - ani~no group dr-ld the . t.i,;os;.i . 5icic ch,arna (31-1

g"C"lp

* 7

Ilrte ultrasonic absorption study carried o u t by !i,~.;,ici~~indrcjti 132j in this

iahwratory in aqueous amino acids of I - ahnine, I - ~~aliine, I -- ieuci~ne, dl - .ila:-nlne ,and

dl - vafine in the pH range 8 - i0.5 and in the trequency range 2 - 30 XIHL ~t t r ibute the

&sc.r\.ed excess absorption can be due to the proton triinsi'er process occurring in these

, I L ~ ~ O L I S amino acids solutions. The excess iiltrasonic absorption obtained arourtd the

neutral pH in aqueous 1 - alanine, 1 - valine, dl - alaninr and dl - valine may be due to

the hydration phenomenon

In order to shed more light on the nature of molecular interactions in aqueous

amino acids, ultrasonic absorption studies are carried out in the acjueous solutions of

jl- alani~ze, 1 - cyestine, 1 - glutamic, dl - seriae and l - tyrosine in the present work.

The ultrasonic absorption studies are carried out in the freq~lency range 3 - 89 MHz.

and at different pH ranges. The pH of aqueous j!?- ula?iine, I - cyestine, dl - swine and

1 - tyrosine solutions are varied in the range 6.3 - 12.6, whereas for aqueous

1 - glutamic solution, it is varied in the range 3.5 - 10.75. The ultrasonic absorption

study carried out in these aqueous amino acids at different concentrations and at an RF

frequency of 10 MHz showed an absorption maximum at chosen concentration.

4.2. RESULTS AND DISCUSSION:

The amino acids @ - alanine, I - cyestine, 1 - glutamic, dl - serine and 1 - tyrosine

are of ARJBDH quality and are used as such without further purification. Aqueous

solutions of p - alanine, 1 - cyestine, 1 - glutamic, dl - serine and 1 - tyrosine are ,I

prepared in the concentration range of 0 . 0 5 ~ ~ ~ ~ ~ using double distilled water. The

reason for choosing this concentration for pH variation can be explained a s follows.

The ultrasonic absorption study carried out on these aqueous amino acids at different

concentrations and at an IW frequency of 10 MHz shows an absorption maximum at

tj-j.s concentration. l'he pH of the aqiieous ari-rini:, ,~cjd+ %>j:~tioni '8r.c ;tdju~',:$~i Ly ,111

E/icLi />H m c i ~ ( E - l i j l j ha~.iilg polasslurri chioric!e g1ai.i eieclrodc to a n ilccuracy ot

B.2pl-i. The pH of the solutions is adjiisted to cfiiferer-it \.aliles by the 'iddition of

krrorvn quantities of standard volumetric solrltion o f I h \aOH sokation.

The ultrasonic absorption st~tdies are carried out in the aqueous soliltions ot

- alanine, 1 - cyestine, I - glutamic, dl - serine and 1 - tvrosine in the frequency range

of 3-89 MHz using Pulsed Pozi1~r Osciiinicr and A4AlrtC 77110 sysfcrn as given in Chapter

11. 'The ultrasonic velocity is measured using an Ui f rasc i~ ic Tii-izc Ilrfer-z'iii0t7z~'t~'r (UTI-102)

by Pulse Echo Overlap Method (PEO) in the frequency oi "a OMMz as disci~ssed in chapter

11. The density and viscosity of the solukions are mecisitred using i'pcc$c Griziiif!j Boitie

and Ostu~ald's Viscometer as given in chapter 11. The temperature of the soli~tions is

maintained at 303 K by circulating water from a thermostatically controlled 5vater bath

with an accuracy of rtO.1K The temperatures of the solution and the circtllated water are

noted by using a dual terminal digital thermometer designed in this laboratory by

inserting them in the holes, provided in the ultrasonic liquid cell.

The ultrasonic velocity data for aqueous solutions of P - alanine, 1 - cyestine,

1 - glutamic, dl - serine and 1 - tyrosine at different pH are given in tables 4.1 to 4.5

respectively. The variation of ultrasonic velocity with pH is shown in figures 4.1 to 4.5

for aqueous p - alanine, 1 - cyestine, 1 - glutamic, dl - serine and I - tyrosine

respectively.

From the value of absorption coefficient (a), the observed absorption ubs

computed. These absorption data are fitted to the conventional Debye type single

relaxation equation (1.16) in the frequency range studied using the non-linear least

square fitting algorithm proposed D. W. Marqudart [33]. The non-linear fitting

program is written in FORTRAN language [32] and is given in appendix A. From the

computation of non-linear fitting program, the relaxation amplitudes A & 8, the

6 70 8 9 10 11 12

pH Figure 4.1 V a r ~ a t ~ o n of u l t rason~c ve loc~ ty

with pH of 0 OSmoldm aqueous p-a ian~ne

pH F igu re 4.3 Variat ion of u l t r a son ic veloci ty

with p H of 0 . 0 5 r n o l d m ' ~ a q u e o u s L-g lu tamtc acid

(I)

," 1518 - -

pH Figure 4.4 Variat ion of ultrasonic velocity

wlth pH of 0.05rnoldm-~ aqueous DL-serine

pH Figure 4.5 Variation of ultrasonic velocity

with pH of 0 . 0 5 r n o l d m ~ % ~ u e o u s L-tyrosine

rt.iaw!i~[~ frequency j; and absorption per .iv,t\.t.!t.rlgfJl ! r~ , i , i ;,rc L , ~ i 1 8 F k l t c e ~ \!i]lere '\ I\

the relaxation amplitude, B is the absorption d ~ i c tc3 :kw ~cri\.cnt !,?a5 <1:72' rcs~dLiI~P

relaxai.i~~~ processes occurring with very high rriaz;?iiiirm !reqtltb~iiies i < r o r i ~ the .rbi,i.r

parameters, the relaxation time z is c')Ii.tii;ated tising the rt.i,ltion,

The maximum absorption per wavelength Id) , IS iaPcitic~tcJ u\ir~g i!~c reliliioii,

I'he computed parameters vlz, relaxatlon freqi~rncy f,, relLjx,ition ,inlpiltridrs

A & B, relaxation time T, absorpt~on per maximum wai elength [ail 9 ,, dnd sol~irne

change of the system Av and kinetic parameters viz. tortvard k , and bnckt~arcl A, rate

constants for aqueous p - alanine, 1 - cyestine, 1 - glutamic, dl - serine and I - tyrosine

respectively at different pH range are given in tables 4.6 to 1.13 and in 4.11.

f a The variation of observed absorption 1 for individual concentration and

[s- ,,,,, the absorption per wavelength (d) as function of frequency at different pH are gi.rre11

graphically in figures 4.6 to 4.20.

Figures 4.6 - 4.15 shows the variation of observed absorption with frequency for

aqueous solutions of p - alanine, 1 - cyestine, 1 - glutamic, dl - serine and 1 - tyrosine

respectively in the pH range studied. The observed absorption decreases with

increasing frequency. The variation of observed absorption with pH increases with

increasing pH values of P - alanine, 1 - cyestine, 1 - glutamic, dl - serine and 1 - tyrosine.

It can also be seen that the values of observed absorption increases in magnitude with

an increase in the p H of P - alanine, 1 - cyestine, 1 - glutamic, dl - serine and 1 - tyrosine

respectively.

f r e q u e n c y [f] [MHz]

' , 7 0

Frequency [q [ M H z ]

Figure 4.6 Variation of observed absorption with frequency for aqueous p-alanine in pH 6-70 and pH 8.30

Frequency [fl [MHz]

3.1 10

Frequency [q [MHz]

Figure 4.7 Variation of observed absorption with frequency for aqueous p-alanine in pH 9.57, pH 10.99 and pH 12.12

10 Frequency [f] [MHz]

Frequency [fj [MHz]

Figure 4.8 Variation of observed absorption with frequency for aqueous L-cyestine in pH 6.41 and pH 8.05

N -* 103

k Z

? 83 0 .7-

X - -z 63 *L a - u

S 43 .- + E n m 23 71

I % 3

8 3.1 10 90

Frequency [fl [MHz]

Figure 4.9 Variation of observed absorption with frequency for aqueous L-cyestine in pH 9.18, pH 10.79 and p H 12.14

Frequency If] [MHz]

10

Frequency [fl [MHz]

Figure 4-10 Variation of observed absorption with frequency for aqueous L-glutamic in pH 3.50 and pH 4.92

10 90

Frequency [g [P;IHz]

i I I 3.1 10 90

Frequency [q [MHz]

Figure 4.11 Variation of observed absorption with frequency for aqueous L-glutamic in pH 6.93, pH 9.30 and pH 10.75

F r e q u e n c y [fl [ M H z ]

F r e q u e n c y [q [MHz]

Figure 4.12 Variation of observed absorption with frequency for aqueous DL-serine in pH 6.35 and pH 8.27

I I 3 1 10 90

Frequency [fj [MHz]

Figure 4.13 Variation of observed absorption with frequency for aqueous DL-serine in pH 10.17, pH 11.86 and pH 12.56

Frequency [MHz]

Frequency M [MHz]

Figure 4.14 Variation of observed absorption with frequency for aqueous L-tyrosine in pH 6.66 and pH 7.94

10 Frequency [fl [MHz]

Figure 4.15 Variation of observed absorption with frequency for aqueous L-tyrosine in pH 9.74, pH 10.00 and pH 10.44

'The variation of absorption per iv,i\.eleng?.Ia (uj-i \vitE.i irli:cij.i:>t; iieijiienib.

[cnli~ilateJ) is @\.en graphically in figures 4.16 1.232 idi 13 ,.n/,!il:i:c, 1 - ci.c5tji-.if,

1 - glutantic, dl - serine and 1 - tyrosine respectivel;. in the pfd r,irli,;e >:uiiicli, f*rL:nl the

figures, i t can be seen that the absorption per ival.eIengtEi for tire aqni.ot:s sclLltiorls of

fi - aianine, 1 - cyestine, 1 - glutarnic, dl - serint. and ! - t~ro:;jne z! L{it"ers.nr values

incresses with increasing frequency and reaches a rnasimui:~ \ c ~ l u e ,3t '1 pjrticul;~r

frequency called relaxation frequency ,L of that particular concentration and then

decreases further with increase in frequencv. For any particui,tr frt.yiiency, the

absorption per wavelength has shown a non-linear variation ~vith ii~creiist' i n pH oi

p - alanine, 1 - cyestine, 1 - glutan~ic, dl - serine and 1 - tyrosine. Also, t!~e relaxation

frequency .L decreases to a lower value with increase in the pH rraiue oi fl - alanine,

1 - cyestine, 1 - glutamic, dl - serine and 1 - tyrosine.

+3 Salient features of the study are szrmrnarized as,

J The ultrasonic velocity increases with increasing pH value tor ~ ~ q u e o u s

p - alanine, 1 - cyestine, 1 - glutamic, dl - serine and I - tyrosine.

J The adiabatic compressibility decreases with increasing pH value for

aqueous p - alanine, I - cyestine, I - glutamic, dl - serine and I - tyrosine

respectively as seen in the ultrasonic velocity profile.

J The free length decreases with increasing pH value for aqueous

j3 - alanine, 1 - cyestine, 1 - glutamic, dl - serine and 1 - tyrosine.

J The density of the solution increases with increasing pH value for

aqueous p - alanine, 1 - cyestine, 1 - glutamic, dl - serine and 1 - tyrosine.

The shear viscosity increases with increasing pH value for aqueous

fi - alanine, 1 - qestine, 1 - glutamic, dl - serine and 1 - tyrosine, but it

initially decreases below the value of solvent distilled water.

8 ,

a 5 *> 'I'he obser1.t.d absorption - OC.~~L>L)-+.-- { V I ~ ~ I ~ I ; L ~ L ~ ~ . ~ * L . in !~L~,.~L!L~;~L-&. tcbr \ t 2 , "

any particular pH i~i1ile of j3 - riiar2i~~e, 1 - c~~c.?!ji~tr, i - r ; i ~ i t , i ~ : ? i ~ , dl -- s e r l r ? e

and I - tyrosine.

3 The value of observed absorption illgedbeb i ~ i t f ~ ~ ~ l c ~ ~ ~ l s e 111 \ . ; ~ ~ I J Q ~t

f~ - alanine, I - cyestine, 1 - gl~~iamls, dl - serine and i - tyri?>lrar. ir-i water

+:+ For any particular pH value, the sarrcttion &pi iibsorptiitn per $\,i\.elei~gth

(d) shows a maximum a t the relaxaijon freijue~~cq. , fr c ~ t that p~rtictllar

pH value for p - alanine, I - cyestine, I - giutl~mlc, dl - ierlne ailci

1 - tyrosine.

-3 The ~palues of absorption per wa.i.eiengthjd) show a ncn-!l~lear variation

with increasing pH value for p - alanine, 1 - cyestine! 1 - giutnmic,

dl - serine and i - tyrosine.

*:* The relaxation frequency A shifts tonpards a lower value with iilcrease in

pH value of p - alanine, 1 - cyestine, 1 - glutarnic, dl - serine and

1 - tyrosine.

+3 The relaxation time t shifts towards a higher value with increase in pH

value of p - alanine, 1 - cyestine, 1 - glutamic, dl - serine and I - tyrosine.

8 The relaxation amplitudes A & B show a non-linear variation in the pH

range studied for fi - alanine, 1 - cyestine, I - glutamic, dl - serine and

1 - tyrosine and relaxation amplitude A shifts towards a higher value with

increase in pH value of P - alanine, 1 - cyestine, 1 - glutamic, dl - serine

and 1 - tyrosine.

From tables 4.1 to 4.5 and figures 4.1 to 4.5, it can be seen that the ultrasonic

velocity u of aqumus amino acids for varying pH values shows an increase with

increase in the pH value for all the amino acids studied. The adiabatic compressibility

.,re,i>es with the ii~crease in the pH .i-,iluei tor drr\ji-io ,)i.ici\ 5:ii,jil,.~ I I~~ , ,tri,llraii of J . -

Ui i , r , l>f i r~ i~ .i,eiocity with increase in p i i i'~iiie il-i;lv b t b cp l i i i3 j -Ei l \L8j i l',i-jsIiil.i'ij bV

rc.+oriing to flickering cluster n-todei of zv,iter.

According to this model, the water is siipposed roi?siit of j:"i.jrt?seJS -Enncfcd

cjiratt.rs and unbonded water molecules. The moleciiies in tile i n t e r i ~ r 01 the c l ~ i ~ t ~ ~ c , are

yuadri~ptiy bonded (ice likej and unbonded :cater moiecuics arc s ~ i ~ ~ o ~ e d t o I?I 'CL~P~V r .

the space in between the clusters. 'The cliisters are sornctiines reierrc.3 ;s:s "pen

structure' water and the dense monomeric fluid is referred to as 'clilhcd stauct~ire'

water. The mixture is a dynamic mixt~lre and the break do.iz.n ui clusiers is a

cooperative process. When one hydrogen bond breaks in the ciuster, tht' ~ s h o i e cluster

breaks do.rkrn resulting of an increase in the close packed structure oi tzrdter. tl'hers the

amino acids are dissolved in water, it splits into two viz., anions and cations a re formed.

The water molecules are attached to the ions strongly by the electrcstatic forces, ~i-hich

introduce greater cohesion in the solution. When NaOH is added, whose net structure

breaking property would first disturb the open structure of water thereby increasing

free monomer population, an action that would be followed by structural

reorganization leaving the molecules in closely fitted helical cavities. Such a n increase

in the closed packed structure of water results in increased cohesion of water molecules

leading to an increase in the ultrasonic velocity.

The increase in ultrasonic velocity of aqueous amino acids is attributed to the

breaking of clusters by OH- molecules resulting in increasing the closed packed

struQure of water. They first would disrupt the water structure, an action which could

be followed by structural reorganization leaving the amino acids in closely fitting

helical cavities. It is known that such an increase in the close packed structure results in

increased cohesion between the water molecules [34]. This increased cohesion decreases

the adiabatic compressibility resulting in an increase in the ultrasonic velocity c as

oit3tj;ved in the p ~ h e ~ " tbt~dy as seen ir? the i i g r i i t . 5 ~ . ~ - ":..?A i j I 1 dl, I.~~.~~~,....; ; t i . 4 ; uIis u!lrLi-~nic 1,etosity is increasing aa the pki \ . i l i i je orairc.i-cci ji:r t i l c , l i j tili. \i.\ti.nl

I t is also knotvn that the incre,isc i;a t l j :rLjiC,lli i < ~Jc;~!;. iicj i l~. i l t l , i l i l l t I1ll

acrii hoi~ttions is due to hydrat~on of the amino z c i ~ i sno!czcul~>i 1%. hlih i:tir.i.iic* ~ ( ~ ~ I ~ ~ I o ~ I

be:v,vren \cater nlo!ecuies.

In the present ultrasonic absorption 3t~tdies on P - ,ti,lrlnnc, E - ivt.s:ir:c',

i - glutamic, dl - serine and 1 - tyrosine solutions, the adtra>oiaic ,~il>t)rptiu~i:, i tSt;~l l lei i 111

the pH range 6.3 - 12.6 for - alanine, 1 - cyestine, dl - serlne a i d 1 - t\.ro:-.i::t) iitiii i r i the

pti range 3.5 - 10.75 for I - glutamic and in the frequency rz;ngr 3 - 83 lz ,Ire tt>unc? to

be greater than that d ~ ~ e to the solvent water. 'I'his indicates that there e\isth some

mechanism by which the observed ultrasonic absorption of riqueoris ammo acids gets

increased. From figures 4.6 to 4.15, it can be been t h ~ t the observed ultrasonic

absorption (5) decreases with increasing fwqnency thereby shor\.ing that the ubs

ultrasonic absorption is frequency dependent. This decrease of ultrasonic absorption

5) with frequency is an indication of the presence of relaxation time 1351 in the .t+ / "h ,

I '\ a I measured frequency range. The experimentally measured values of absorption - 1 Lf - are plotted as open circles in the figures 4.6 to 4.15. These experimental points fit very

well to the equation for a single relaxation as indicated by the continuous line in figures

4.6 to 4.15 and thereby giving the information that all amino acids studied show a single

relaxation behavior in the pH range studied and in the frequency range 3 to 89 MHz.

From the figures 4.16 to 4.20, it can be seen that for all the amino acids studied,

the value of absorption per wavelength (d) increases with increasing pH of the

aqueous amino acid solutions. Also, the relaxation frequency .f, of an amino acid

solution shifts towards a lower frequency value with increasing pH of the aqueous

1 - ' I ' ' ' . . I

3.1 10 90

Frequency [q [MHz] Figure 4.1 7 Variation of absorptiog per wavelength

ld mL with frequency for O.O5~%qeuous L-cyestine

3.1 10 Frequency [fl [MHz]

Figure 4.1 8 Variation of absgrption pe r wavelength with frequency for 0.05~!i~^e:ous L-glutamic acid

10

Frequency [fl [MHz]

Figure 4.1 9 Variation of per wavelength with frequency for DL-serine

15

14

13

12

11

10

9

8 -4- pH7.94 -b- pH9.74

7 -+ pH1O.OO -+ pH10.44

6 3.1 10 90

Frequency [fl [MHz]

Figure 4.20 Variation of absorption per wavelength d -2

with frequency for 0 . 0 5 ~ ~ ~ u o u s L-tyrosine

chapter Iv ,21ninu Ac1d5.. . - amino acid solutions. From the above results, it is more probable that the proton

transfer process may be considered as the principal source for the obser\red excess

in aqueous solutions of P - alanine, 1 - cyestine, dl - serine and 1 - tyrosine in

the pH range 6.3 - 12.6 and 1 - glutamic acid in the pH range 3.5 - 10.75 and in the

frequer~cy range 3 - 89 MHz. The above results are also in good agreement with the

theory for proton transfer proposed by Hussey and Edmonds [29].

A similar situation of excess absorption, increase in the value of (ai),, and the

shift of relaxation frequency towards low frequency side with increasing pH has

been reported by Holmes et a1 [36] in aqueous glycyltyrosine and cysteine solutions in

the physiological pH range. They have attributed this behavior to inter- and intra-

molecular proton transfer in these solutions. Hence the above explanation holds good

for the present excess absorption observed in aqueous P - alanine, I - cyestine,

1 - glutamic, dl - serine and 1 - tyrosine solutions at different pH.

The results of the present ultrasonic absorption studies on aqueous amino acids

at different pH are further supported by the ultrasonic investigation of Hussey and

Edmonds [26] on aqueous solutions of protein and polypeptides. According to Hussey

and Edmonds, in addition to the proton transfer process other mechanisms such as

interaction between the solute and solvent molecules, the perturbation of salvation

equilibria, conformational changes keto-en01 equilibria may have their contribution

superimposed on those due to the proton transfer to the excess ultrasonic absorption

below pH 8. The amino acid molecules in the neutral solution generally exist in dipolar

form, thus have a stronger interaction with the surrounding water molecules, i.e.

hydration [lo]. This hydration may be the cause for the observed excess absorption

around the neutral pH of aqueous solutions P,- alanine, 1 - cyestine, 1 - glutamic,

dl - serine and 1 - tyrosine.

From the table 4.11, it can be seen that the computed ~,alues of \ .ol~~rne change of

the system 4 v and kinetic parameters v~z. forward k , and backward k, rate constants

for aqueous f3 - alanine, 1 - cyestine, 1 - glutamic, dl - serine and 1 - tyrosine respecti~~ely

are well within the reported values of other amino acid studies where proton transfer

mechanism plays a dominant role in the pH range of Hussey and Edmonds 1261 and

Applegate et a1 [37].

4.3. CONLCUSION:

The ultrasonic absorption studies carried out in the frequency range 3 - 89 MHz

in aqueous solutions p - alanine, 1 - cyestine, 1 - glutamic, dl - serine and 1 - tyrosine at

different pH show that the ultrasonic absorption decreases with increasing frequency

and increasing with increasing value of pH. The absorption variation follows a single

relaxational behavior. The possible relaxation mechanism can be due to the proton

transfer reaction process occurring in these aqueous amino acid solutions. The excess

ultrasonic absorption obtained around the neutral pH in aqueous P - alanine,

1 - cyestine, 1 - glutamic, dl - serine and I - tyrosine may be due to the hydration

phenomenon. Kinetic parameters computed supports the relaxation mechanism is due

to proton transfer reaction in aqueous amino acid solutions.

Table 4.1 Ultrasonic velocity and related parameters for aqueous ,f? -alanine

x = pH of 0.05ndd.lp-alanine; p = density; qs = shear viscosity; u = velocity;

ps = adiabatic compressibility; = classical absorption; Lf = free length;

Table 4.2 Ultrasonic velocity and related parameters for aqueous L-cyestine

12.14 1013.53 0.8348 1519.67 4.272 6.177 0.412

x = pH of 0.05!n.*-?~estine; p = density; qs = shear viscosity; u = velocity; -

pS = adiabatic compressibility; = classical absorption; Lr = free length;

Table 4.3 Ultrasonic velocity and related parameters for aqueous L-glutamic acid

10.75 1009.41 0.8242 1519.72 4.289 6.122 0.413

x = pH of 0.05'ildC~lutamic acid; p = density; qs = shear viscosity; u = velocity;

pS = adiabatic compressibility; = classical absorption; Li = free length;

Table 4.4 Ultrasonic velocity and related parameters for aqueous DL-serine

- x = pH of 0.05h~e~-ser ine; p = density; qs = shear viscosity; u = velocity;

pS = adiabatic compressibility; = classical absorption; L,f = free length;

Table 4.5 Ultrasonic velocity and related parameters for aqueous L-tyrosine

x = pH of 0.0&-<~rosine; p = density; T+ = shear viscosity; u = velocity;

p s = adiabatic compressibility; = classical absorption; LF = free length;

Table 4.6 -3

Ultrasonic absorption and related parameters for

MHz Npm-Isz Npm-Is2 s x 10"

6.70 17.04 60.28 1.265 0.934 7.794

12.12 12.38 73.09 2.536 1.286 6.882

x = concentration of amino acid; f r = relaxation frequency; A & B = relaxation amplitudes; s = relaxation time; (ah)rax = absorption per maximum wavelength;

Table 4.7 -3

Ultrasonic absorption and related parameters for 0.05in.f"a*~ueous L-cyestine

pH f, A B T (ck!A)maY

MHZ N P ~ - = S ~ ~ p m - l s ~ s x 104

6.41 18.02 65.23 1.268 0.883 8.911

12.14 12.24 75.68 2.254 1.301 7.038

x = concentration of amino acid; fr = relaxation frequency; A & B = relaxation amplitudes; z = relaxation time; ( a ? b ) ~ ~ = absorption per maximum wavelength;

Table 4.8 - 3 Ultrasonic absorption and related parameters for 0.05 r n ~ k d *

aqueous L-dutamic acid

MHZ ~ p m - l s ~ ~ p m - l s ' s x l o4

x = concentration of amino acid; f r = relaxation frequency; A & B = relaxation amplitudes; z = relaxation time; (ah)3Ta, = absorption per maximum wavelength;

Table 4.9 -3

Ultrasonic absorption and related parameters for 0,05~%"queous DL-serine

pH fr A B % (ah)mm

x 10-l5 x 10-l5 x

MHZ ~ p m - l s ~ ~ p m - ~ s ~ s x l o4

6.35 17.24 59.36 1.105 0.923 7.764

8.27 16.18 63.15 1.365 0.984 7.756

10.17 15.02 64.89 1.597 1.060 7.402

11.86 14.74 66.15 1.959 1.080 7.409

12.56 12.31 68.13 2.158 1.293 6.377

x = concentration of amino acid; 6 = relaxation frequency; A & B = relaxation amplitudes; z = relaxation time; (Oih)max = absorption per maximum wavelength;

Table 4.10 -3

fara Ultrasonic absorption and related parameters for 0.05r"' aqueous L-tyrosine

pH f r A B 'C (crh)max

MHZ ~ p m - l s ~ ~ p m - l ~ ~ s x lo4

6.66 17.94 60.25 1.198 0.887 8.203

x = concentration of amino acid; f r = relaxation frequency; A & B = relaxation amplitudes; z = relaxation time; ( a h ) m n \ = absorption per maximum wavelength;

Table 4.11 Kinetic parameters for various aqueous amino acids

Amino acid k kb Av

concentration) m-lS-l s-I m3/mol

ki = forward rate constant; kt, = backward rate constant; A v = volume change;

Ctiapt~r Iv 5 l i n l t : ~ Aczdc.. .

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