BIOCHEMICAL

Vol.68,No.4,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

STRUCTURAL PROPERTIES OF MOJAVE TOXIN OF CROTALUS SCUTULATUS (MOJAVE RATTLESNAKE) DETERMINED BY LASER RAMAN SPECTROSCOPY Anthony T. Tu Department of Biochemistry Colorado State University Fort Collins, Colorado 80523 and B. Prescott,

C. H. Chou and G. J. Thomas, Department of Chemistry Southeastern Massachusetts University North Dartmouth, Massachusetts 02747

Received

December 16,

Jr.

1975 Summary

Laser Raman Spectra were obtained on aqueous and solid samples of Mojave toxin isolated from the venom of the Mojave rattlesnake (Crotalus scutulatus). __-.The Raman spectra reveal that the Mojave toxin, an acidic protein of molecular weight about 22,000, contains a predominantly a-helical secondary structure and that the tyrosyl residues, on the basis of the Raman frequencies and These features of the Mojave toxin intensities, are exposed to the solvent. distinguish it structurally from the neurotoxins of sea snake venoms. However, like the sea snake venom toxins, Mojave toxin contains four disulfide bridges and is not greatly altered in structure by removal of the aqueous solvent. Rattlesnakes snake

found

of the genuses

in the

Americas.

species

of rattlesnakes

species,

the Mojave

identified

the

rattlesnake genus

of its

isolated

Bieber

(isoelectric

chemical

neurotoxins greater

mentioned than

(1)

isolated

designating

krait,

it

is

States

the most

major alone,

toxic

toxin

are all

9 and molecular

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

venoms

lethal

toxin,"

properties.

venomous eighteen

so far

(2),

toxin

weight

smaller, weights

1139

of about basic

from

and reported

a

Unlike

the

neurotoxins

Mojave

toxin

is a

was shown to be an acidic

and molecular

above

principal

"Mojave

and sea snake

Mojave

of 4.7

the

The venom of one of these

the

and toxicological

Moreover, point

United

(C. scutulatus),

et al.

from cobra,

cardiotoxin.

continental

are

Crotalus.

venom of C. scutulatus,,

number

points

In the

and --Sistrurus

have been categorized.

among the

Recently,

Crotalus

protein

22,000),

proteins

of 7000 to 8000)

whereas

(isoelectric (2).

Mojave

the

Vol.68,No.4,1976

toxin

also

venoms

(3,

BIOCHEMICAL

differs

chemically

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

both

from

the

4, 5) and from

crotoxin

(6,

containing

acidic

as basic

protein

undertaken

to determine

the neurotoxins

as well

The conformations (10)

venom toxins

Therefore, with

differences

from

sea snake

toxin

as previously established

among the

by acrylamide-gel

the 514.5

disc

nm line

native

study

structurally

was

from

sea snake by laser

reported

and cobra

Raman spectroscopy.

here,

10) to identify

(9)

can be compared

specific

structural

toxins.

the venom of Mojave of the

electrophoresis, toxin

of an argon-ion

differs

Crotalus

mixture

The present

including

The homogeneity

(1).

due to the

also

toxin, (9,

from

a neurotoxic

previously

Raman data

described

presumably

(8),

of Mojave

was isolated

is

from other

venoms.

have been investigated

published

isolated

components.

toxin

of many proteins

or similarities

Mojave

band,

Mojave

the Raman spectra

previously

7) which

whether

isolated

protein

laser

rattlesnake

toxin

preparation

which

(Coherent

was

indicated

Raman spectra

(1).

and purified

Radiation,

were Model

a single excited

with

CR 2) and

In Water

1700 1660 1566 Figure

1.

1400 1366 1266

Raman spectrum

at 32“C of Mojave

venom (400-1700 Conditions: spectral set;

1100 ~C$QQ 900 cm

Aqueous

cm-l). excitation

slit

amplification

width

wavelength

toxin solution 514.5

10 cm-1 ; scan speed 1.

1140

800 isolated

700

600

500

!

from C. scutulatus

at pH 6.5, nm; radiant 25 cm-'/min;

74 mg toxin/ml. power rise

300 mw time

10

Vol.68,No.4,1976

BIOCHEMICAL

recorded

on a Spex Ramalog

handling

for

are

to an accuracy

Raman spectra

Details

spectrometer.

Raman spectroscopy

reported

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are

given

elsewhere

of the Mojave

toxin

in aqueous

are shown

in Figs.

1 and 2, respectively.

amide

I line

centered

at 1650 cm -1 , with

on the

high

a clearly

frequency

1251 cm-1 .

near

920-980

cm-l,

stretching

Fairly

with

peptide of the

backbone

conformation

scattering

above

is

consistent

ination

Figure

strong

with

2.

1 with

I and amide

amide

occur

the

band.

(Fig.

l),

the

In the

amide

III

region,

-1 with

weaker

Raman scattering

also

occurs

in the

III

indication toxin

that

of model

interval

to skeletal

(12,

1274 cm-1 suggest

the

extent.

toxin

radiant

1141

1650 and 1274 cm-

This

power

that

Raman

random-

interpretation

through

the exam-

of known conformations

isolated

powder.

a-helical

The weaker

further

and proteins

Lyophilized that

13).

have been established

compounds

cm-').

by the

of a predominantly

protein

at 32°C of Mojave

exception

regions

to a much lesser

correlations

venom (400-1700

broad

solid

of a weak shoulder

at 935 cm-1 , attributable

1650 cm-1 and below

Raman spectrum

and in the

the possibility

Raman scattering

in the Mojave

of Raman spectra

solution

In H20 solution

at 1274 cm

is a clear

@structures

The Raman frequencies

backbone.

respectively,

and/or

rather

a peak centered

of the

frequencies,

of this

peak appears

The dominance

chain

side

discernible

(11).

and sample-

of f 2 cm-'.

state

is

of instrumentation

from C. scutulatus Conditions: is

150 mw.

as in Figure

Vol.68,No.4,

(8,

1976

12, 13).

previously

BIOCHEMICAL

Strong

5 cm"

a medium-strong difficult

interesting

structure

from

protein

in the Raman spectra predominantly In the positions

solid

(12).

clearly eliminated.

than

spectra,

and 2).

These

conformation

and sharp

scopy.

2 the high Fig.

III

amide

is

again

is therefore, non-a-helical

It

also

of an a-helical

to those

observed

believed

to be

for

in the

The absence

shoulder

are

found

at 509 cm-'

(Fig.

of all

four

disulfide

among them,

they

are

not

disulfide

to amide

I is more

liquid

water

baselines

has been the

(cf.

in the

same

Figs.

1

protein

(ii)

in the

2500-2600

to four

are

region

are

of sulfhydryl

disulfide

a high

similar.

to be detected

stretching

1142

toxin

The appearance

indicates

enough

of the Mojave

vibrations

S-H.

linkages large

of a-helical

change

Raman spectra

line

and weaker

solvent.

1) assignable

of this

2) the

to be nearly

the different

to S-H stretching of cysteinyl

(Fig.

presence

from

of Raman scattering

the absence

of the

the

interference regions

toxin

(1266 cm-'

confirm

frequency

of the aqueous

shape

III

at most a marginal

be attributed

The position

It

but

conformations.

of Mojave

and amide

1, since

features

and sytnnetrical

geometries exist

line

cm")

indicate

noteworthy

indicates

disordered

1665 cm -'

indications

which

1665 f

near

residual

identical

protein

near

Random-chain

13). the

spectral

powder)

compensating

results

(i)

(12,

qualitatively

Raman lines

the

after

the following: cm-1 that could

I line

I line

whether

the

coat

I (1656

upon removal

Other

that

are

amide

in B or random-chain

(lyophilized

in

However,

in both

sharp

sample

In Fig.

visible

amide

identified

(14).

at 1247 cm")

structure

a strong

been

19).

1235 f 10 cm-1 .

near

are

of a viral

of the amide

scattering

This

toxin

a-helical

(18,

and sharp

the Raman data

however,

in the Mojave

has also

at 1248 + 5 cm-'

backbone

to note,

a strong

give

line

935 cm-'

structures

line

hand,

III

amide

of the

III

other

to ascertain

regions

give

amide

on the

g

of a-helical

generally

and a strong

structures,

is

Raman scattering

as a feature

B-structures

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

frequency

groups.

of a strong linkages.

probability If geometrical

The

that

the

differences

by Raman spectroat 509 cm-1 in the

Vol. 68, No. 4, 1976

spectrum

BIOCHEMICAL

of the solution

to correlations

506 cm-1 in that

(or

a gauche-gauche-gauche

configuration

established

of the

by Sugeta

of many prominent

Raman lines

The lines

831 and 852 cm-'

at 645,

residues

of the

0.8:l.O.

Since

model tyrosyl are

toxin

groups

(lo),

analyses

to the

for

present

shown in

present 622,

of a sharp

1005,

are

also

the

relatively

of the toxin

(iv)

(Figs.

(1).

could

For the most

the

solution

is

part,

previously the

fact

reported that

cm"

amide

(not

toxin

I and amide

toxins

predominantly

conformation

it

is obvious

tryptophan

is

cannot

be

at higher

the tryptophan

solvent

(17). to the

at 1209,

(c) three

The lines

in the regions reveal

at

phenylalanine

above aromatic residue

III

thus

exhibit

of neurotoxins

in Fig.

amino

present

acids.

in the

400-1700

cm-l

little

or no changes

a number

of dissimilarities

frequencies whereas

B-conformations. 1143

(9,

10).

of Mojave amide

frequencies

1.

resolution

residues

1586 and 1616 cm"

of the

shown)

(l),

1

acid

(20).

Raman spectra

a-helical

resolved

that

Raman lines

in Fig.

in the Raman spectrum

are clearly

states

of Mojave

a predominantly indicate

all

protein

no amino

of tryptophan

to the one histidine

and solid

The Raman spectra

Although

of phenylalanine

the

toxin

of the

1417 and 1552 cm-'

line

of several

in

solvated")

surface

that

to the

of

in the Mojave

in the Raman spectrum

(d) The lines

be assigned

residues

toxin

suggests

tyrosyl

intensities

on the

-1

(a)

ratio

"completely

of the toxin.

1011 cm-'

acids.

to the five

cm"

(i.e.,

1356,

1 are assigned

frequencies

The appearance

amino

on the outer

1011,

line

in Fig.

828:853

further

according

an intensity

obtained

exposed

1 and 2) and 2800-3100

between

from

(The

peak at 1356 cm

due to coincident

toxin.

lines

(iii)

assigned

tyrosyl

located

two frequencies

and 1033 cm-'

No Raman lines

the

881,

1005 cm-'

1 are

suggests

networks,

16).

of exposed

previously

toxin.

(15,

the

residues

of these

1 but

are

residues

were

from the Fig.

The lack

tryptophan

in the

distinguished

for

that

at 757,

C-C-S-S-C-C

two exhibit

and probably

tryptophan

the appearance

indeed

appears

solid)

to the aromatic

in Fig.

indicative

(b) The lines

assigned

from

it

exposed

molecule.

assignable

The latter

is

of the

et al,

of 0.71:l.O

Gly-Tyr

relatively

are

(1).

a ration

compound

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Most toxin

important indicate

of the neuro-

1

Vol.68,No.4,

A second and the lines

BIOCHEMICAL

1976

striking is

found

due to vibrations

toxins

with

corresponding intensities

indicate

that

five

in the

virtually

the

presence

than

is

af adsorbed

tyrosines

tyrosyl

residues

o-helical

with

on the

toxin

structure,

neurotoxins

secondary

since Fig.

surface

of the

entirely

while

the

is consistent

on the

residue

1.

are

could

protein

has

be due to

or to interactions of "exposed"

with

its

of a "buried"

their

presumably

doublet

The concept

hypothesis

average

in each of the

solvent

This

consistent

with

and probably

the tyrosine

groups.

is

The different

are

and the

2 as in

hydrophilic

in Mojave

secondary in the

other

tyrosine

neuro-

at 834 and 846 cm-'

significant

tyrosyl

at 831

In the

(9).

toxin

of

tyrosine

0.8:l.O.

are

of Mojave

as well,

in Fig.

from

1.2:l.O

toxin

intensities

reported

two studies

between

water

ratio

the single

solid

lines

were

ratio

residues

same appearance

of the

predominantly

predominantly tyrosyl antiparallel-

structures.

The present

results

conformational

logical

in the

tyrosyl

lyophilized

the

vibrations

The interactions

mimicked

in

of the Mojave

and relative

We observe

in the

to solvent

neurotoxins.

determine

venoms these

Raman spectra

frequencies

intensities

observed

the

more accessible

B-sheet

in the

intensities

relative

group

between

of tyrosine.

relative

of sea snake

with

the

difference

neurotoxins

and 852 cm-'

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

suggest

properties whether

specific

the

of other

importance

of isolating

cardiotoxins

secondary

and comparing

and neurotoxins

structures

are

in order

prerequisites

for

to

toxico-

specificity. ACKNOWLEDGEMENTS This

to A.T.T. Grant

work

was supported

ay N.I.H.

to G.J.T.

grant We also

by N.I.H. AI11855 thank

grants

to G.J.T.

Peter

2ROl GM 15591

and 5ROl GM 19172

and by a Research

Canny for

isolating

Mojave

Corporation toxin.

REFERENCES 1.

2. i:

5.

A.L. Bieber, A.T. Tu, Ann. C.A. Benin C.A. Bonilla, J.W. Dubnoff (A. de Vires Science Pub.,

T. Tu, and A.T. Tu, Biochim. Bio h s. Acta, 400, 178 (1975). Rev. Biochem., 42, 23573 I--=. -andM.mo, Jr Chromatogr., 5&, 253 (1971). Anal. Biochem.,-32-, 522 (1969). and F.E. Russell, in Toxins of Animal and Plant Origin and E.Kochva, eds] Vol. 1, pp 361-367, Gordon and Breach New York (1971). 1144

VoL68,No.

6. 7. 8. 1:: 11. 2: 14. ii: 1;: 19. 20.

4, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

R.A. Hendon and H. Fraenkel-Conrat, Proc. Nat. Acad. Sci. USA, 68-, 1560 (1971). J. Horst, R.A. Hendon, and H. Fraenkmonrat,Biochenophys,. Res-. m., 46, 1042 (1972). T.G. SpGo, in Chemical and Biochemical A lications of Lasers, Volume I (C.B. Moore, Ecimefi' Press, N.Y., +%qT--N.T. Yu, T.S. Lin, and A.T. Tu, J. Biol. Chem., 250, 1782 (1975). N.T. Yu, B.H. Jo, and D.C. O'Shea, Arch. Biochemxo h s., 156, 71 (1973). G.J. Thomas, Jr. in Vibrational S ectra and Structure*ume ,-&. 3 (J. Durig, Ed.), Marcel Dekker, Inc., N.Y., M.C. Chen and R.C. Lord, J. Am. Chem. z. 96, 3038-4750 (1974). T-J. Yu, J.L. Lippert and W.r Petl'colas, B%i'ol mers 12., 2161 (1973). G.J. Thomas, Jr. and P. Murphy, Science, l88, 1205 197q. * H. Sugeta, A. Go, and T. Miyazawa, Chem. Letters, 83, (1972). H. Sugeta, A. Go, and T. Miyazawa, Bull. Chem. xc. Japan, 46-, 3407 (1973). N.T. Yu, J. Am. m. sot., !3&, 466419m B.G. Frusliourand J.L. Koenig, Biopolymers, 13, 1809 (1974). P.C. Painter and J.L. Koenig, Biopolymers, 14, 457 (1975). A _c?mplete list of the observed Raman frequencies of Mojave toxin (400-3100 cm ) with assignments to amino acid residues is available from the authors on request.

1145