Differentiation of the tyrosyl groups of ribonuclease A by iodination

Vol. 5, No. 1,1961

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DIFFERENTIATION OF THE TYROSYLGROUPSOF RIBONUCLEASEA BY IODINATION* Chul-Yung

Cha and Harold A. Scheraga

Department of Chemistry, Received April

Cornell

Ithaca,

N. Y.

19, 1961

Three of the six tyrosyl normal (Shugar, sequence of this offers

University,

residues

1952; Tanford protein

the possibility

of rlbonuclease

et al, 1955).

IS known (Hlrs of locating

normal groups in the polypeptlde matlon about the conformation

Since the amino acid

et al, l%O),

the positions chain,

are ab-

thereby

of the molecule

iodination

of the three providing

ab-

lnfor-

In solution.

EXPERIMENTAL Rlbonuclease the recommendations lodotyrosyl Several

A was lodinated

of Hughes and Straessle

groups without

derivatives

at 10°C at pH 9.4 according

affecting

were prepared

other

to

(1950) to produce diamino acid residues.

with different

degrees of lodl-

nation.++ RESULTS Alkaline carried

Titration

Data:-

out at alkaline

Spectrophotometric

titrations

were

pH at 312 w and 295 ml.l (1O'C) to deter-

mine the number of dllodotyrosyl

and tyrosyl

residues

(Tgble

I).

*This work was supported by research grant No. E-1473 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, Public Health Service, and by grant No. G-14792 from the National Science Foundation. **The tyrosyl groups react with iodine at different rates; three groups react rapidly and the remaining ones more slowly. For example, three groups reacted completely In 1.5 hrs. The fourth group was not completely lodinated after 3 hrs. When an amount of Iodine equivalent to six tyrosyl groups was added, two groutis remained un-lodinated even after 25 hrs., the iodine color persisting for this length of time. 67

Vol. 5, No. 1,1961

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I TITRATION OF DIIODOTYROSYL GROUPS Number of tyrosyl groups equivalent to the amount of iodine added

Derlvatlve A

Change in molar extin;tiz ;;;ff$clent,

Number of tyrosyls

iodlnated*

,

pK*+

15,000

3.1

8.0

B

3 4

18,000

3.8

7.8

C

6

18,800

3.9

7.6

*Based on a value of 4800 er single group of dilodotyroslne in insulin (Qruen et al, 1959P . **The observed pK1s are somewhat higher than that of dilodotyrosine, 6.5, (Qruen et al, 1959) and close to that of monoiodotyrosine, 8.2, (Herrlott, 1948). However, the appearance of the U.V. absorption The high pK1s spectrum indicates that monolodotyroslne is absent. may be due to local Interactions in the lodlnated protein. The titration

at 295 R&Lshowed an increase

3000 between pH 8 and 12.5 In derivatives this

contrlbutlon

of the un-iodinated

At pH 12 and

the abnormal tyrosyl

on standing Is similar

12.5

at room temperature to that of native

U.V. Difference obtained

A, B, and C.

is due to the dilodotyrosyl

to the Ionization

for

in E of about groups rather

abnormal tyrosyl groups ionize

several

groups. Irreversibly

hours.

This behavior

U.V. difference

spectra were

at low pH and compared with those for native 1961).

than

rlbonuclease.

Spectra at Low pH:-

(Hermans and Scheraga,

However,

The difference

spectra

ribonuclease of deriva-

tives A, B, C are similar to each other and to that of the unlodlnated molecule. Since the native protein contains two tyrosylcarboxyl

hydrogen bonds imbedded In hydrophobic

and Scheraga, exist In all

1961),

In derivatives 3 derivatives;

regions

it appears that these interactions A, B, and C. it

Precipitation

seems that the solubillty

Is decreased at low pH by lodinatlon. 68

(Hermans still

occurs below pH 1 of ribonuclease

BIOCHEMICAL

Vol. 5, No. 1,196l

U.V. Absorption tives

Spectra:-

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The U.V. abeorption

spectra

of deriva-

A and C were examined between 270 w and 3aO w at pH 10.5

and 11.5, respectively, 305 w according tyrosinee

to search, for monolodotyrosine

to Herrlott,

(peak at

1948) and the un-iodinated,

abnormal

(peak at 276 W, since the abnormal tyroeines

be protonated

at these PIXIE).

Two peaks were observed,

mg and one between X0 w and 311 m&. The former un-iotinated

tyrosyl

C) and the latter 4 in derivative

to dilodotyroayl

curves for tyrosyl peak will

groups (3 In derivative

C).

A,and

In derivative

2

group8 (3 In derivative

It can be shown, by addition and dliodotyroayl

sorption

ape&rum.

one at 287

is due to the

groups,

that the tyroeyl

16 can also be concluded

curves that monolodotyroaine

A and

of separate

appear at 287 w because of the contribution

dliodotyrosyl

would still

from the

from these ab-

la absent.

CONCLUSIONS 1.

Two tyroayl

conditions

groups of rlbonuclease

used here (derivatives

iodlnated

tyrosyl

are not lodlnated

B and C).

one of the abnormal tyroeyl

(derivatives

B and C), but at a slower rate.

glnce derivatives

pounds have un-iodinated ference Ionize

spectrum must arise irreversibly

spectrum,

abnormal tyrosyl

ribonuclease

and since all groups,

from two of the tyrosyl

at alkaline

A).

groups can be iodlnated

A, B, and C, and native

the same low pH U.V. difference

the

rapidly

groups are the three normal ones (derivative

In addition,

2.

The three

under

have these com-

the low pH dlfgroups which

pH.

3. Since one of the abnormal tyrosyl groups can be iodlnated without changing the environment of the other two abnormal tyrosyl groups,

the lodinated

one, but with high pK.

abnormal group may be a reversibly Presumably, 69

it

ionizable

cannot be dletlngulshed

Vol. 5, No. 1,196l

BIOCHEMICAL

from the two other

abnormal groups In an alkaline

native

molecule

because of the irreversible

mation of the native

molecule

to conclude that this tyrosyls,

Is not near a carboxyl

titrates

possible tyrosyl 4.

unlike

like

titration

of the

change in the confor-

at high pH.

tyrosyl,

to the U.V. difference it

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

It Is also reasonable

the other

group since it

two abnormal doee not contribute

spectnun at low PH. When It is iodlnated

the three

other dliodotyrosyls,

small conformational

suggesting

change in the vicinity

a

of this

upon lodlnatlon.

The conformations

molecule

of derivatives

appear to be slmllar

Enzymat$c hydrolysis the 3 and 2 un-lodlnated, between dllodotyroslne Similar

methylated

rlbonuclease

experiments

have abnormal tyrosyls. are In progress

abnormal tyrosyl

experiments

if

the distinction

any of the latter

are being carried

(Broomfleld

to locate

groups of derivatives

should permit

and monoiodotyroslne,

fy the abnormal carboxyl abnormal tyrosyl

since all

Such studies

A and B, respectively. Is present.

A, B, C and the native

and Scheraga,

groups which are Interacting

out with 1961) to ldentiwith the

groups. REFERENCES

Broomfield,

C. A., and Scheraga, H. A., Unpublished work (1961). Gruen, L., Laskowski, Jr. M. and Scheraga, Ii. A., J. Blol. Chem. e, 2050 (1959).

Hermans, J., Jr. (1961)

and Scheraga, H. A., J. Am. Chem. Sot.,

In press

l

R. M., J. Gen. Physlol., 2, 19 (1948). Him, C. H. W. Moore, S. and Stein, W. H., J. Blol. Chem., a, 633 (ld. HugheTig$)". Jr., and Straessle, R., J. Am. Chem. Sot., 72, 452

Herrlott,

Shugar, D., Blochem. J., 52, 142 (1952). Tanford, II,

C., Hauensteln, 6409 (19%) .

J. D. and Ftands, D. G,, J. Am. Chem. Sot., 70