CHAPTER VII

ORD AND NEW DISPERSION RELATION

 194

ABSTRACT

A general introduction is given to Optical rotatory

dispersion and the theories of ORD are presented. As
an attempt to form an alternative technique to study ORD,
a new expression from dispersion relation is developed
and is applied to the study ORD in simple and bio-molecules.
The results are discussed critically in relation to the experi­
mental values obtained and earlier reported data and the
feasibility and the success of this low cost instrumentation
for the study of ORD are outlined.
 195

7.1 INTRODUCTION :

The phenomenon of changes in optical activity

with wavelength of a polarized beam of light has

been known; since the negining of the nineteenth

century. Here, the poineering work of Biot1, Fresnel2

3
and Pasteur may be considered as the base of modern
1 2
stereochemistry. Biot and Fresnel showed that

the rotatary power of an optically active medium

increase with the decreasing wavelength of the incident

light. Only in 1930's, the phenomenon of optical

rotatary dispersion (ORD) took on a new turn due

4 5 6 7
to the studies of Tschugaeff , Rupe , Kuhn , Lowry ,
Mitchel^ and Levene and Rothen^. Later Haidinger11^

observed the differences in absorption of the component

of circularly polarized light. This phenomenon is

known as circular dichroism (CD).

The technical difficulties on the applications

of ORD and CD to organic chemical problems were over

come only in the 1950's when a photo-electric spectro

polarimeter became available. The improvements in

instrumentation were immediately followed by numerous

applications of ORD and CD techniques to organic

chemical problems. Among the dynamic and productive

groups working in these field the main are the groups

of Djerassi11 and Klyne12.
 196

Optical rotatary dispersion study of organic

11 13-17
molecules ' has been used m determination

of absolute configurations and relative positions

of fundamental group; in analytical problems such

as the separation of racemic mixtures; in the detection

of weak absorption bands (magnetically allowed, electri­

cally forbiden) or bands which are poorly resolved,

in the stereochemistry of complex formation; and

finally, in solvation problems and the study of confor­

mational equilibria with temperature. ORD has also

developed into a powerful tool for the characterization

of the conformations of bio-molecules, mainly in

estimating the types and contents of secondary structure

of proteins nucleic acids and polymers^ to ORD

is also used to determine base stacking characteristics.

Recently ORD technique is used in medical field 23 ' 24

for Diognosis especially in Diabetes. Finally magnetic

ORD (MORD) and magnetic CD (MCD) open up the possibility

of inducing optical activity in any molecule (Faraday

effect).

ORD and CD have certain advantages even

over the X-ray crystallographic studies as the former

are based on solution techniques requiring absolute

minimum perturbation to the system which means that

experimental conditions can be chosen so that physio­

logical relevance is ensured and further more in

 197

the solution state, desired perturbations such as

change in concentration (of protein or any other

component, such as substrate salt, effector, or denatu-

rant) can be effected more readily than in the crysta­

lline state.

7.2 THEORY :

Optical rotation and circular dichroism

represent two inseparable aspects of optical activity

which result from the interaction of a given medium

will linearly polarized light. Linear polarized

light is regarded as a super position of two equal

left and right circularly polarized components fig.(7.1).

Optically active media are characterized by different

indices of refraction for the two components, nR

/ nR, and (since n = c/v, c = velocity of light in

vaccum) different propagation velocities vR ^ vR

as well as different angular velocities of the projec­

tion of electric field vectors fig.(7.1) passing

through a material medium of unit path length. One

component gains a lead over the other and remains

fixed after the light has left the optically active

medium. Hence, the light continues to be linearly

polarized but the plane of polarization is now rotated

at angle of

... (7.1)
<=k ■=■
(degr)
 198

Pig.7.1 : Projection of the electric field vectors

showing composition of linearly
polarized light

(a) Two equal components of left and right

circularly polarized light
(b) The situation after light at a
wavelength which is not absorbed
has left an optically active medium
 199

Where ^ is the vaccum wavelength of the

light beam.

Specific rotation expressed dts property

of materials depends on the pathlength (in decimeters)

and concentration (in g cm-3 ). It is defined as

7, 25, 26.

r* degr cm^
c- L \ g. dm ... (7.2)

with , and to a less extent

with temperature (T) and concentration (C).

The term molar rotations [ Q ] takes into

V

account the molecular weight (M) of the solute, and

is expressed as

r degr.cm'
z"' ” \oo
1U decimole

For Biopolymers, the data are usually expres­

1 "• <7‘
3)

sed in terms of mean residue dotations.

1 Us
> \bo
degr.cm
... (7.4)
decimole

Where Mq is the mean residue molecular weight.

Mo of a protein can bedetermined from its amino

27
acid composition. Moffitt introduced the term

reduced mean residue rotation [ vv\ i ]^

,T by including
>
lorentz field correction.
 200

degr. cm
(7.5)
decimole

Where ’ n' is the refractive index of the sol­

vent at the specified wavelength.

7.2.1 Drude Equation :

2.8
'
Drude expressed a relationship between

optical rotatary power and wavelength outside the

region of an optically active absorption band.

M>= '<■ /) ... (7.6)

Where and K are the dispersion and rotatary

coefficients. Equation (7.6) is called one term

Drude equation. This equation is experimentally

, 7
validated by many investigators, notably Lowry .

One term Drude equation is obeyed in most of the

X
proteins in the visible reason. A plot of [vd,] X

versus [m‘ 3^ yields straight line with 2 as the slope

and K as the intercept.

Two term Drude Equation :

A two form Prude1 r, equation i:: found I i>

be applicable to most cases and the equation reads

as

... (7.7)
 201

Where the first term expresses the rotatary

contribution of a chromophore with an absorption

maximum at/V^ and the second term refers- to the contribu­

tion of a second optically active absorption band

at ^2* ai an<^ a2 are constants of the complex

dispersion curve. [ Equation (7.7)]. The dispersion

curve is still monotonic just as in one-term Drude

equation. If they (a^ and a2 > are of opposite sign

and further J a-^ [ > | a2 | and^-^ <A2 an anam°l°U3

dispersion can be anticipated i.e., the curve passes

through a maximum or minimum, crosses over the zero-

rotation axis, and changes the sign of the rotation

at certain wavelength.

On rearranging, the equation {1 . 1 ) can

be rewritten as

(7.8

The four parameters ar a^ and *2 can

be evaluated by plotting a graph between l\}~ '^

viruses

7.2.2 Mofitt's Equation s

ORD curve of purely helecal forms cannot

be explained by one-term Drude equation. In this

29
regard Moffitt theoritically developed an expression

as
 20/

[W] -ia''x'P'h3 l^-Mi

..(7.9)
)v A

Where Ai is the dispersion constant, and

gi and b. are two other constants. Of these b.

1 1 is
specifically related to the helical content of the

macromolecules. Some objection has been raised by

30 31
Kauzman and by Murakami regarding ■this form of

equation.

Since equation (7.9) is not directly applica­

ble to the treatment of experimental data, Moffitt

20
and Yang modified it to the simplified form.

(7.10)
\ I

Still it has three adjustable parameters

a°' b° andOxjzpT yield

a straight line. The slope and intercept of the

resulting straight line will give b0 and aQ respectively.

b0 refers only to the measure of helical

content. In the absence of helical structure b0

equals to zero and Moffitt equation reduces tp the

Drude equation. For proteins and polypeptides,

Apis estimated as 212 + 2 nm.

7.3 ORD FROM NEW DISPERSION RELATION :

The new dispersion relation of Murthy et

32 .
al is modified by rearranging the terms, as
 203

..(7.11)

Here n is the refractive index and (| is

the slope of dispersion curve (the graph is plotted

for y ^-against l/(n-l).

Moffitt's equation (eq. 7.10) for mean

residue rotation rewritten as

> (7.12)

substituting the value of W x~ 0 ^ froin

On
' Ao
eq. 7.11) in the above equation (7.12) one gets the

equation for mean residue rotation as

ai Oo-O bo

> . QL©
/
bo (7.13)
Q.o ¥

In the above equation (7.13), a0 and b0 are constants

characteristic of molecular rotation.

7.4 EXPERIMENTAL SET OP :

The polarimeter is used to rotate the plane

of polarization to reestablish an original null

condition either by movement of the analyzer or by em~

33
ploying the Faraday effect . It is useful to study

the optical rotation of the system in visible region,

and is depicted in the fig. (7.2).

 204

Fig.7.2 : Experimental set up for ORD

-T\ 205

A light beam of a powerfull source (sodium,

Mercury and Cadmium) is made to pass through the

spectrograph and the sample, which is placed in the

polarimeter cell. Here the constant deviation spectro­

graph with a symmetrical attachment acts as a monochro­

mator. This is supplied by Toshnieval and Brothers,

Madras, and the polarimeter is procured from ASCO

India. The cell (Asco make) used in this set up,

is the same as that used for polarimetry. The lamps

used are of low pressure type supplied by scientific

syndicate Hyderabad.

To start with, light beam from a monochroma toi­

ls passed through the polarimeter (empty cell), the

analyzer and the reading on the analyzer is noted

for minimum intensity position, then the sample is

introduced in the cell. Then again the analyzer

of the polarimeter is set for the minimum intensity

position and the reading is noted. The difference

between the two readings correspond to the optical

rotation of the system in circular degree. The procedure

is repeated at various wavelengths by adjusting the

drum of the constant deviation spectrograph.

7.5 RESULTS AND DISCUSSION :

Equation (7.13) for ORD from new dispersion

relation is applied to a few systems viz., d-Glucose

 206

BSA {Bovine Serum Albumin) and Insulin. They are

procured from M/s. Aldrich, USA, M/s. Sigma, USA

and M/s. Boots Company, India respectively. The

solutions are prepared by using suitable solvents

to required concentrations and PH values, which were

mentioned in the references 34-36. While preparing

i
*

the solutions, care is taken about the suspended

particles, which produce the scattered light, and

effects in the study of ORD. It is, therefore, most

important to remove even small amount of precipitate

either by ultracentrifuge or from the filteration.

Refractive indices at various wavelengths,

needed to study the ORD, for 0.32N d-Glucose, are

determined in the laboratory by using Pulfrich refracto-

meter (Asco make) reading to an accuracy 1 in 10

and are found to agree with the values reported in

references (34) and are listed in table (7.1). The

values of [m']^ calculated from equation (7.13) are

compared with the [m*values obtained from equation

(7.10) and with standard [m'] values. There are

also given in the table (7.1). ' The values a , b

obtained by Moffitt's equation as well as equation

(7.13) are also listed in the table (.7.1). The values

and needed in the equation (7.13) are determined

from the graph plotted l/\2 against l/(n-l) and are

given in the table (7,1).

 207

ORD of BSA in water at PH 5.7 and Insulin in 3 M

urea at PH values 8.5 and 3.5 are studied. Refractive

indices of three samples at various wavelength are

determined by Pulfrich refractometer and are listed

in the tables (7.2) to (7.4), and are evaluated

from the graphs drawn between 1/^2 and l/(n-l) and

are also reported in their respective tables. The

values a , b and X are taken from the references

o o ' o
(37), (38) and (39) and the/\Q values are determined

from new dispersion relation. They are also given

in the respective tables [Tables (7.2) to (7.4)].

A glance at the tables (7.1) to (7.4) showed

that the [m']^ values obtained from' equation (7.13)

and (7.10) and experimental study are fairly giving

the good agreement between them. The values of [.M„

obtained from equation (7.13) are close to the [m1 ]

values of Moffitt's expression (7.10), whereas the

experimental values are not much closer to the Moffitt's

values. The ORD curves are drawn between wavelength

versus [m‘]^ values obtained by both methods and

experimental methods. Prom the observation of the

graphs 7.1 to 7.4 it is also observed that there

is the near markedness between [m' ] values determined

from equation (7.13) and (7.10) for all samples.

The deviation in the experimental values is due to

the poor sensitivity of the experimental set up used.

The use of photo electrical detection along 5 with

 208

sensitive recorders might reduce the deviations in

the value.

Thus, the applicability of new dispersion relation

to the study of ORD in case of simple molecules and

biomolecules results in a good success.

The results also show the effective use of

refractometry in lieu of spectropolarimeter assembly

for the study of ORD. Thus the function of refractro-

metry as a 2-in one instrumentation is set beyond

doubt.

The merits of the present approach to the ORD

over that of Moffitt's are :

(1) Moffitt's approach is empirical whereas the

present one is based on rigorous algebraic formula­

tion.

(2) The relation adhoc fixation of value of(Mofitt

equation) is now superseded by the exact determina­

tion of the same without leaving scope for an;,

arbitrariness. can be determined without

ambiguity from the new dispersion relation.

The values of/\D obtained in BSA. Insulin (at

two PH's) by new dispersion relation are found to

be very close to the value of 212 nm as taken arbitraily

by Moffitt.
 209

The last and foremost important aspect of the

present investigation is the success in suggesting

a low cost instrumentation for ORD in lieu of 0031:15/

spectropolarimeters (of VARIAN make or others).

 Table 7.1 : ORD of D-Glucose 0.32 N, Water
Refractive B —2
index 46.6667 x 10 cm [m 1]. degrees-cm 2per
A decimole
m ti
■n*
2.50x10 , From o = 965.5 A c From
0.0753x10' Moffitt's ac 2.53 x 10' N.D
expression ^ O-OS, * 10* rela­
tion

From Moffitt's . Standard

From eq.
(7.13) eq. (7.10)

r*
404. 1.4552 141.58 140.38 137.07

o
479. 1.4530 121.36 119.38 118.84
01*901
104.51

o
508. 1.4515 104.27

92.87

o
535. 1.4504 94.41 92.70

75.16

o
589. 1.4485 76.22 74.77

59.34

o
656. 1.4400 60.11 59.05
210
 T a b le 7 .2 : ORD o f B o v in e serum a lb u m in H o t PH 5 .4

1
f\l

00
R e f r a c tiv e

II
'
E

E
O
H

r--
i—i
f-
A in d e x d e g re e s -c m 2 p e rd e c im o le

00 1

O li
= 212 nm fro m

^
M o f f i t t 's e x p r e s s io i

o o
X 00

oo• (30
nm
•n*

<H
= 2 1 1 .8 5 nm fro m N .D . r e l a t i o n
i
i

i
i
ii

ii
ii
ii

ii
i
i

i
ii
t
i

i
i

i
i
i
i

31
i
i
i
ii

1
1
11

1
11

oo 1
1
o 1
1

1
1
II 1
1
1 1

43 Dl
1
11
From e q . From e q . E x p e ri­
(7 .1 3 ) (7 .1 0 ) , m e n ta l

1
CM
rH
r^
rH
—

i—1
4 0 4 .7 .5 3 5 0 -1 7 2 .9 3 1 8 3 . 05
CO

1—1
o
00
in•

so
rH

r-*
00

sr
.4 9 6 0 -1 0 9 .5 4 -1 2 6 . 24
CTii

00
in

5 0 8 .5 .4 7 8 1 -8 6 .1 7 -8 5 .2 1
00

rH
5 4 6 .1 .4 6 5 0 -7 0 .3 6 -7 0 .0 9 -7 5 .

1
o
o

to

rH
5 7 9 .0 .4 5 5 6 -6 0 .0 7 -6 6 . 45

H
5 8 9 .3 .4 5 3 0 -5 7 .9 1 -5 7 .9 3 -6 3 . 72

rH
6 4 3 .8 .4 4 1 1 -4 6 .1 9 -4 5 .9 3 -5 9 . 97
211
 Table 7.3 : ORD of Insulin PH 8.5, 8M Urea

Refractive

00
-2 2

t
II
pH

<
o
u
e

0*
CM
X
co

00

cj
index . [m 13 degrees -
-cm perdecimole
nm

e
u
0

a
a = -692 = 212 nm Moffitt's expression
'
Ao
o

fl

II
Q

e
•

C
it
M
0

CM
rH
co
£

A
o
relation

From eq. From eq. Experi­

(7.13) (7.10) mental

r-
rH
404.7 1.5545 -262.56 -261. -269.53
00
to
467.8 1.5099 -180.70 -178. -187.82
'

i
o

rH
in

in
CO
«A

508.5 1.4896 -146.79 -145.

(Tv

546.1 1.4747 -123.44 -122. -134.22

rH

579.0 1.4640 -107.34 -107. -115.47

r^
00

589.3 1.4612 -102.99 -102. -112.52

CO
"3*
rH
in

643.8 1.4474 -83.70 -95.23

to
H*
to
 Table 7.4 : ORD of Insulin PH 3.5.8M Urea

Refractive

t
00
2

1
-2

II
X
o
—i

oo
C\
CO
00
<J\

QJ

A
index cm [in'K degrees -cm. per decimole

c
e
ii
212 nm f.*om Moffitt's expression

d
= -510
o

o
u
.
D relation
1
1
1

1
1
1

I1

1
1
1

z 1
1
1
1
1
1

1
1

tfc-4 1]
0 1
e 1
1
1

1
c 1
E 11

r—I 11
CM• 11

it 1
CN 1
1

l
Dl

CO 1
1
1
1
1
1
l
l

1
XI 1
31
II 11
1
1 1
From eq. From eq . Experi­
(7.13) (7.10) mental

404.7 1.5589 -203.42 -201.46 -218.93

467.8 1.5110 -137.31 -135.82 -144.75

96*601-
508.5 1.4921 -111.25 -121.25

546.1 1.4751 -92.79 -92.39 -104.06

579.0 1.-4654 -80.64 -80.39 -90.72

CM

i—1
r-'
i£>

*
589.3 -77.34 -77.14 -85.12

643.8 1.4506 -62.90 -62.91 -70.24

213
 0 Standard
X Eqn. 7.10
4 Eqn. 7.13
/ Sectm ote ■
£W 3/\ d e g r e e s -

400 4$t> sroo 55© 6oQ 65t>

A nwt

Graph 7.1 : ORD curves of d-glucose

 f

ZOt) ~

0 Expt.lT
* Eqn. 7.13
„/ Eqn. 7.10

i9>

o
£
‘3
0-
IS

VJ
t
wv
<S>
5,
If
"G

j,
5-0

------ 1-------- 1-------- 1-------- 1-------- 1-------- 1---

400 4-5-0 fTO D 5^0 5c 5 tfr*>
^ /VA
Eraph. 7.2 : ORD curves of BSA
 4",

-cvn' / dt?u mol<z

A <Ae<jpees>

Graph 7.3 ORD curves of Insu^in PH 8.5

 4
/;■-."

Graph 7.4 : ORD curves of Insulin PH 3.5

 22

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 A NEW DISPERSION RELATION; RELATION TO ORD, MORD and MOLECULAR POLARIZATION

V. Rama Murthy, H, Jeevan Kumar and D. V. Subbaiah

Molecular Bio-Physical Laboratories, Department of Physics

Sri Krishnadevaraya University, Anantapur 515 003 AP India

polarizability (Molar polarization) are

ABSTRACT derived and applied to monomeric, oligo­
meric and polymeric states successfully.
A new dispersion relation developed This will enable the utility of refracti-
for the first time is applied to get vity as a 3 in 1 instrumentation for
(i) an expression for mean residue stereochemical analysis of sinple as well
rotation (of ORD), (ii) an expression as synthetic and bio molecules. The
for magneto-optic rotation (of MORD) details of the method are presented below.
and (iii) mean molecular polarizability
of molecules. These methods enable Method
any stereochemist to study the con­ Starting from the fundamentals, the
formations of any simple as well as relations for the dispersion is given
synthetic and biomolecules by sinple as [l]
determination of refractive indices at
various wavelengths. This will enable
1 «• 2n£-JL [.
2. 2 ] (1 )
the utility of refractive technique as
'(u‘ , 2* .
a j in 1 instrumentation for attempting o , + b
a systematic study of sterochemical ' n
analysis of biomolecules as well as But since<-»= 2n’i = 2sc/x and
any other type of molecules.
•*»0 = 2«c/x0* a further rearrangement
and si nplifi cation yields
1/^ *
•e
INTRODUCTION Tn-TJ

Natural dispersion, optical

rotation dispersion and magneto optic whpre a 1/ x0 and
rotatory dispersion are the three
important optical phenomena, which are e2 N
used very much by physicists and stereo-
chemists. Though these phenomena are
P - - 2nC2m
the results of response of an electron
(free electron) to the incident light The details of derivation of eqn.(l)
beam in natural, asymmetric and is given ref.[2]
magnetically induced asymmetric enviro­
nments respectively, a possible correl­ ORD:
ation among these is not envisaged and a
survey of literature in this direction The Moffit's equation for reduced
resulted in limited success. So the mean residue rotation is given as
authors have taken up this aspect of
study and the results of the present r i•> . ao V * bo *o
investigations reveal a definite im J a 7-5--------- 37 * T
progress in this direction. <A - agz) U3— grg
- V)
Startin'* from the fundamentals, a
r.ew dispersion relation is derived. The , - [aG/{ a2/xd2 - 1) ♦ bo{A2/^ 1}2]
irr?portancg of thi.r ffilation is
by the fact that by using this
expressions for (a) mean residue where SQ is the wavelength corresponding
rotation {of ORD) (b) magneto-optic
rotation (MORD) and (c) mean molecular to natural frequencies of electron, Ms
the wavelength of source of radiation and

1636—IEEE/£ighth Annual Conference of the Engineering m Medicine and Biology Society

.-o.hh. |; . ,.fTV:, >. tr-i
aQ, bQ are constants characteristic of one can get
molecular conformation.
( \ „ r
But one can write from eqn.(l)

1 _ (n-x)
“ <?#■ A substitution of eqn.(6) in Loventz-
Ti- ' Lorenz relation yields

On substituting this value in Moffit’s

equation, equation (2) results M (7)
P (a-p )*

a'pCn-l) , (2) N, M and refer to Avagadro number,

molecular weight and density while a
and p are the intercept and slope of
linear dispersion relation eqn.(1)
bo respectively.
where a0*
RESULTS AND DISCUSSION
In the above equation, aQ, bQ are
The equations (2), (4) and (7)
constants characteristic of molecular yielding expressions for m1- (of ORD),
conformation. (of MORD) and aM (of molecular polari­
MORD: zation) are applied to several molecular
systems. While 0.32 N d-glucose is taken
From eqn.(l) n can be written as as example for ORD, benzene and PEG 400
are taken examples for MORD and benzene,
n ' xylene, CS2, NaCl, KC1 CaF2 and PEG 400
Then (!-■<>?-)
LDu±>\ ±!C2ti>X (3) are taken as examples for molecular
polarization. The [aD] for 0.32 N glucose
n (i *f)
where is taken from reference [4] and refractive
, . P A indices of glucose at various wavelengths
f ‘ 1-h Ab­
are determined in the laboratory using
Pulfrich refractometer (of ASC0 make)
reading upto ♦ 0.0001. The refractive
using this value of (n2 - l)2/n in indices and [m*] calculsfted and the
values of constants aQ, bQ obtained by
the equation for magneto optic rotation
of Murthy and Naidu [3], one gets an Moffit*s equation as well as eqn (2) are
expression for MORD as reported in Table 1. The values of a and
p needed in the equation (2) are obtained
n
from the graph of 1/^ vs l/(n-l).
c _ Kft rI U *4 )
o
(7,
V ’ \
/

The magneto optic rotation as

where K is a constant (= 7i/2eN),n pand p
refer to the molecular weight density of
the molecule and number of free electrons
prevalent in the molecule respectively.
A is the wavelength of source of
radiation.
Mean molecular
A case of particular interest arises
when A-*"- Then eqn.(l) reduces to
P (1 - J)
0 ’ 0 + th-t-it or n«
where refers to the refractive index
at infinite wavelength. From eqn.(5)
calculated using equation (4) alongwith
the one determined through Murthy and
Naidu*s expression, calculated for benzene
and PEG 400 are reported in Table II.
The values of the constants like N,M,M
etc. needed in the calculation are taken
from ref.[5] while p, the number of free
electrons is calculated based on the
criteria given by Rao and Murthy [6j.
The reported values are taken from
ref.[7,8].
The mean molecular polarizabilities
of benzene, xylene, CS2, NaCl, KC1, CaP2
(5) and PEG 400 calculated using eqn.(7)
alongwith the reported values [9,10] are
given in Table III. The refractive
indices for these molecules except
PEC 400 are taken from ref.[11] while

lEEE/Eighth Annual Conference of the Engineering in Medicine and Biology Society—1637

TABLE I - ORD (in d-glucose)

Wavelength ["-*3 ' aQ x 10“^ bQ x 10~5

n
(/O 2
degrees cm
(in nm) per decimol. Eqn.(2) Hoffitt Eqn.(2) Moffitt

656 59.34 1.4460 2-53 2.5 0.08 0.0753

589 75.16 1.4485
535 92.87 1.4504
508 104.51 1.4575
479 118.84 1.4530
447 137.07 1.4552

TABLE II - MORD

Wavelength Benzene (£in 10** rats/gauss/cm) PEG ( £ in 10A mts/gauss/cm

(A )
(in nm) Calc. Rep.[8] Calc. Rep.[7]

310.0 160.30 174.279 _

330.6 130.80 138.551 - -
361.1 99.17 102.587 - -
404.1 73.69 75.144 - -
435.8 60.75 — 32.06 30.10
452.9 55.12 55.791 - -
467.8 50.85 — - *
480.0 47.74 — - -
508.6 41.50 * - -
546.1 35-09 - 21.22 19.60
578.0 30.76 - 19.01 17-80
589.3 29.44 30.600 18.70 17.70
643.8 24.10 4“* —

TABLE III - Mean molecular polarizabilities (x 1025 CC)

Compound aM aM aM

(Calc.) (Rep.) (R * M)[6]

Benzene 101.04 103.29 102.12

Xylene 137.02 136.I10 136.92
CS2<C 78.10 87-49 82.10
Rocksalt 32.810
31.82 39-78
CaF? 24.9710 27.61
23.97
KC1 41.31 41.7910 42.87
PEG 400 401.50 392.007

1638—lEEE/Eightfc An'-jal Conference of the Engineering in Medicine and Biology Society

for PEC 400, data is taken from rt*f.[y].
A glance at Table I suggests that
ao» *>0 values as calculated by Moffit's
expression tallies completely with the
one obtained from eqn. (2 ) . This suggests
the validity of the new relationship
between natural dispersion and ORD. The
one advantage of the present method is
that >,0 is not assigned an adhoc value
[like in earlier cases of application of
0RDj[l2j but its value is obtained from
linear dispersion relation without any
ambiguity. This concept of unambiguous
value of helps in conformal analysis
of macro molecules.
The magneto optic rotation values
given in Table II shows a very good
agreement between calculated and earlier
reported values and hence lends very
good support to the relation between
natural dispersions and rtORD.
The mean molecular polarizability
as calculated by eqn.(7) agrees fairly
well with the earlier reported values
and thereby suggests for general appli­
cations of the relation between natural
dispersion and molecular polarization.
One more additional advantage of eqn.(7)
is that the evaluation of noo needs no
approximations while either Cauchy's
relation or Sellimeir* s relation [a]
are only extrapolations of n values at
two or four wavelengths respectively.
Thus the results of present investi­
gations suggest beyond doubt the multi-
ferious utilities of the technique of
refractivity. A thorough determination
of refractive indices at various
wavelengths using either Pulfrich
refractometer (or its modifications
to suit the needs) yields information on
ORD, MORD and molecular polarization •
thereby making refractivity as a 3 in 1
technique wliich finds potential appli­
cation in bio-instrumentation for study
of conformations of biomolecules.
Acknowledgements:
The authors thank the organisers
of Eighth Annual Conference of IEEE/
EMBS, FORTWORTH, Texas (USA) for
accepting the paper for presentation
and encouraging.
lEEE/Eighth Annual Conference of the Engineering in Medicine and Biology Society—1639
REFERENCES
1. I. Fabelinski, 'Molecular scattering
of light', Plenum Publishers, London
1969.
2. Murthy, V.R., Jeevan Kumar, R.,
Subbaiah, D.V. and Sreeramulu, A.,
J. Chem. Soc. (Communicated).
3. Murthy, V.R. and Naidu. S.V.,
Curr. Sci. 48(7), 292 (1979).
4. International Critical Tables II
McGraw-Hill Book Conpany Inc.,
New York and London, p. 347-
5. CRC Handbook of Chemistry and
Physics, 59th edition, 1978-79,
Rubber edition, RC Press Inc.,
Florida.
6. Rao, B.P. and Murthy, V.R., Proc.
All India Syrap. Phys. Res. ft Edu.
Part II, 345 (1974).
7. Naidu, S.V., Ph.D. Thesis,
SV University, Tirupati, India, 1979-
8. Murthy, V.R. and Rangaswamy, Y.C.,
Curr. Sci., 48, 988 (1979).
9. LeFevre, R.J.W. and LeFevre, C.G.,
Rev. Pure 6 Appl. Chem., 5(4), 261
(1955).
10. Batsanov, S.S., Refractometry t,
Chemical structure, translated by
P.D. Sutton, Consultance Bearue,
New York, 1961.
11. International Critical Tables,
Vol. VII, p. 20, 1931, McGraw-Hill
Book Coup any Inc., New York and
London.
12. Leach, S.J., 'Physical principles
and techniques of Protein Chemistry*,
Part C, 1973, Acadamic Press.
 PRE-PRINT - SK UNIVERSITY JOURNAL 1987.

SPlECTHGSCOPlC STUDIES ON a FEW CO-ENZYMES*

V. RAMA MURTHY and R. JEEVAN KUMAR

ABSTRACT
: l

The parallel (£ocn p), non-bonded „) and perpendicular components (%2cc/^

of polarizabilities and mean polarizability (ccM) of a few coenzymes [FMN, FAD,
S-Adenosine metheonioe and o:-Lipcic acid] arc evaluated quantum mechanically.
The diamagnetic susceptibilities and molecular elector) ionization cross-sectionS ar*
also evaluated from the mean molecular polarizabilities. The variation in electron
ionization cross-section is discussed and the need to probe further in this direction is
stressed.
INTRODUCTION
Proteins, nucleic acids and f^ysaccharides form the basic rudiments through
/•/ /wic!/ many life mechanisms like metabolism, growth etc., are explained and the
/ whtcHy
understanding of these mechanisms in terms of chemical activities of these
biomoiccules is gaining importatj'^e now-a-days. Molcc^/lur biclcgy and biomedical
engineering are two fields of research of much importance in this direction. Though
many spcctrdscopic techniques like UV, Visible, 1R, Raman and NMR have bien
used to study life mechanisms in terms of biopolymcric activities, not much work
has been done in utilisirg molecular polarizabiliy as tool in this field. Rao et uL
(1977) and Murthy et al. (1979-1980) have used successfully the technique of
molecular polarizability in the conformations of biopolymers, synthetic p^ymers
end liquid crystals. This paper reports our study of molecular polarizability of few
coenzymes by using Lippincot function potential model and ;usc the same to
evaluate diamagnetic susceptibilities (xm) and molecular etectron-ionization cross
section (Q) and to correlate the Q values with the oxidation potential of the
coenzymes. The reason for choosing only these coenzymes and vitamins is the
following : Of the many enzymes,^some are/some are used -to oxidise or reduce, some
for transferring amino groups, decarboxylation, a.cylation etc., One enzyme under
each category is taken and the reactivity of that coenzym^f over the substrate iu-
terms of specposcopioparameters is studied. s
//
Presented at the DAE Symposium on ‘Biophysicai aspects of Cell stuclure and Function
Held at Lucknow in July, 19S6.
 2

THE METHOD
The Lippincott — 8—function potential mode! is based on the Quantum
mechanical approach to the study of eigenstate of the system and polarizability
forms one of the fine critertion for testing suitability of such wave functions. It
gives an easy and faitly reliable method for the polarizability detcimir.ation.
Relevant equations to calculate the mean polarizability in terms of the parallel
bond component (v or,f erpcndicnlar bond contribution (H 2 and non-bond /-i-/
N
region electron conttibution {t oc,, „) are given as

OCn = £ [s cr.,1, + v xII „ + S 2 cc^J

where
A/
(X,-X2)]' /£* f>/
oc„ p = 4n A Ra + . ... (2)
A X ?j
a„ . 4 2Cr2 4 J

Here n is the bond order; A, the 8 —function strength or reduced

electronegativityy^^the bond length \jx\/tLn$Jklf,the Pauling electronegativities of /X I /
/,«/ atoms A and B in the bond AB. The values of delta function strengths A-’s and
Cr’s, defined by Lippincot and stutman (1965) for various bonds are taken from Refs.
(Sanyal, N. K., Dixit and Pandey, A. N. 1973 ; Rao, B. P., Murtbyjty, V. R, and //
Subbaiah, D. V. 1976). For the second term on the right-hand side of equation io y
we have

S °C|| n = Si fj <xj -(3)

I hi Her^f^is the fraction of the non^oadecFelectrons of the j1* atom and ct; is / b'
its atomic polarizability.

For the third term in equation (i)

-2
•-A Xia
A} oc,-.
W-i-i

S 2 oc. Udf hr**-] -(4)

i i

Here n is the number of degrees of freedom given by the equation nd( = (3N-2t^k,) 6/
/iTtz *y where N is the number of atoms and ns is/thet^umber of bonds in the coenzyme.

The mean polarizabilities of the coenzymes. Flavine mono nucleotide (FMN),

Flavine Adenosine dinucleotide (FAD) S-Adenosine methionine and cc-Lipoic acid
are evaluated by equation (1) to (4).. The necessary data on bond lengths of various
bonds present in these coenzymes is taken from CRC Handbook (1979). The value:; / h I
Ji // Situ a an^ S 2 of
®f-£3u »> 2HI' IPkjdilobg/with
f /7J are given in Table 1.
 *3

diamagnetic susceptibilities and molecular electron ionization cross section.

Though various emperical methods <Guy <?t a!. 1954, Bandit, 1961, Habberditzl,
J964) have been developed to calculate the diamagneti. susceptibility, they were not
found>^o have universal application and hence Rao and Murthy (1979) have suggested
a relation which states that

/W. - xm se fEfm / , -■<(5) / °< H /

/ *-/ Where^=» (0.9,* represents the saturation'factor'' with^deroting the number / -n/
of unsaturated bonds or rings present in the molecule and a is the degree of covalenc-y
of the characteristic gfoup in the molecule, m = 0.72 X 1$The details of this / \<\ /
method are-given in ref. (Rao & Murthy (1979).
This method is used to evaluate xm from the/ghyvalues obtained for coenzyraes j <X M
|>y Lippincott/c^functicn method. The values of xm are reported in Table 2.

m Molpcpiar-electron ionization cross section :

jr\( /if It is well kjfer^wn that there is no porous theory to explain molecular electron
(onization cross section (Fasman, G. D. 1952). Therefore several semi-emperical
relations have been suggested to explain the experimental observation. Electron
ionization cross-sections have been suggested to be proportional to molecular
j. j polarizability as well as diamagnetic suscepiibility./^Beran and Kevan/^1969) and /
' ' ^ao and Murthy (1979) are among those who have proposed semi-emperical relations,
Rao and h^urthy’s relation reads as
/ r*/
Q (in 10">6 cm*) = 0.278,s/b/xh •(6)

hi where/g|/i$ the saturation factor explained in last section. This formula (eq. 6}
is applied to obtain the ‘Q’ values of the coenzymes. The values are given in Tabled.
The oxidation potential taken from Ref. (Fasman, G. D. 1952} of these eoenzymes lav
also given in Table 2 for comparison.
Results and discussion:
Frdm Tables l and 2, it is seen that the molecular polarizability as well as
diamagnetic susceptibility obey additive property as in any other type of simple
/ as/- molecules as well/jbiomolecules. Also the molar polarizability show an Increase with
increass in effective electro-negative character of the group transferied by the
ltd hi //enzyme. VA graph^rdrawn between electron ionization cross section as well aa-
oxidation potential of the, enzyme show a regular variation thereby showing that the
/ :)h/
ionization ability as well as oxidation, potential are well related tbroq^ the number
/to/ ^ree c^ectrons (available for reaction) present in th^fenzymes.
The relatively low values-of* molecular electron ionization cross-section in
/tj FMN or FAD is reflected in its/^ase to oxit^se or reduce a biomolecul^In Lipoid
HI

A
•J'
 tfcid the val\ie is large. Tliis suggests tUc necessary foie it plays-in oxidative-
decarboxylation, A'lso Eipoic add acts as acyl transferring as well as hydrogen-
transferring agent. Similarly the multiple ways of reactivity of adenosine methiorine-
in /(l)/the transfer of sulphur to the t/^borr chain (in serine) as well as (ii)‘
/{O/ /cl/
transmethylation is indicated by the high value of Q as well as^-E Investigations-
in this field requiring' more confirmative approach to the enzyme kinetics are in
progress.

TABLE—f
POLARIZABILITIES ( X !D»* am? )

Co-enzyme S CC|, „ S T--\ „

/ / S 2 7l/

FMN 9.4888* 0 474 30 2.236 4 066.

PAD 15-9991 0.859 58 /4 953 / 7.26X
oc- lipoic acid 4.8930 0.322: 2* 1.969 2.395
S-adenosyl metheo-nine 7.7050 O'436- 38- 2 864 3.668

TABLE-2
^’OLARIZAMLFfrEs/(10a»)/ DIAMAGNETIC SUSCEPTIBILITIES (X10*) AND
MOLECULAR ELECTRON IONIZATION CROSS SECTION AND
OXIDATION POTENTIAL

/W “Zm q
*V(V>
; . Mot. Wl
Coenzyme tfXlp^cc)
rtSS) (xl0,*c“*>

Pmn 4.066 /45 072 / 4.854? -0.211 465.416

Pad 7.26? 106.796 8.385 -0-219 785.900

\j,j /ax/-Lipoic acid 2.395 92.186 20.759 -0.290 216 312

/x-i-CS/ S-Adenosyl metheo-nine y 3 66s/ 103.927 18.774 -0.262 39V.406

REFERENCES :
Bandit, J., J. Cheat. Phy. Physiole. €heffl. Biof. 58, 288, (1961).

Beran, J. A., and Kevan, L., J. I'hys. Chem. 73, 3860, (1969).
CRC Hand Book of physics and Chemistry, 59th ed. (1979),
Fasman, G. D., Hand Book of Biochemistry & Molecular Biology (ed), (1952);
Guy, J., Tillieu, J., Compt. Rend, 239, 1203, (1954).
Habbarditzl, U., Sitzchev. Deut, Akad Wiss. Berlin Ke Chem. Geol. Biol.
No. 2. (1964).
Murthy, V. R , In<f.*l?IhClSem. & Biophys. 16, 32, (1979).

Murthy, V. R., Naidu, S. V., Ranga Reddy, R. N. V., M- & Liq,

Cryst. 59. 27, (1980).
Murthy, V. R., (1978). Subbaiah, D. V„ Naidu, S. V., J. Quant, Spectr. &
Rad. Trans. 19, 551, (1978).
Rao, B. P., Murthy, V. R., Subbaiah, D. V., Ind. J. Biochem & Biophys. 14,
181,(197®.

Rao, B. P., Murthy, V, R., Subbaiah, D. V., Naidu, S. V., Acta Cienica Indies,
5 (p), 118, (1978).
Lippineot, E. R., Stutman, J. M., J, Phy. Chem. 68, 2465, (1965).
Sanyal, N. K„ Dixit, Paridey., A. N., J. Phys. Chem. 77. 2552, (1973).