The removal of testosterone binding globulin from plasma by affinity chromatography

263 THE REMOVAL OF TESTOSTERONE BINDING GLOBULIN FROM PLASMA BY AFFINITY

CHROMATOGRAPHY

Sumner H. Burstein Department of Biophysics,

The Weizmann Institute of Science, Rehovot, Israel”

Received June 26, 1969 ABSTRACT The use of steroid immobilized on an insoluble polysaccharide for the separation of a specific binding protein from human plasma is described. An androstane derivative covalently bound to agarose was effective in removing testosterone binding globulin from plasma. The protein could subsequently be separated from the reagent in low yield by means of guanidine.

The presence in human plasma of a protein which specifically binds testosterone has been indicated by the work of several groups . ’ A facile method for separating proteins, which reversibly bind small molecules from complex mixtures has been known for some time. ’ This method, called affinity chromatography,

has been applied to the purification of enzymes and

antibodies with considerable success.

Experiments designed to separate

estradiol receptor proteins from uterine preparations by affinity chromatography have also recently been published. 3 Unfortunately, these latter authors were unable to obtain the protein free from the specific adsorbent which obviously limits the utility of their findings.

It was thought that suitable adapta-

tion of the above techniques could be applied to the separation of testosterone binding globulin (TBG) from plasma. In general the method involves the covalent attachment of the small molecule to an insoluble polymer in such a fashion that it is still free to bind to its specific protein.

When a mixture containing the specific binding protein

On leave from the Worcester Foundation for Experimental Biology, Shrewsbury, Mass.

264

STEROIDS

14:3

is passed through a column of such a polymer, will be retained.

Subsequent displacement

only the protein which binds

of the protein

should yield a

pure substance. The polymeric purified

saccharide

beaded form

is available

commercially

in a

which has excellent chromatographic 4 A method has been described for linking primary-amino

properties. containing

agarose

molecules

(Sepharose)

to agarose

by means of cyanogen

nature of the bonding is not known, however,

bromide.

it is believed

The exact

that a urethan 2

type derivative decided

Based on the experience

is formed.

that an agarose

complex

of others

would give the best chances

it was for success

in

this case. The problem sterone

affecting

In fact the essential

binding affinity.

only a 17 (3-hydroxyl

p-01 is commercially

available

The binding constant which was considered chromatography.

therefore,

and seemed

of TBG is reported

sufficiently

no interference

shown to be a

3 P-Aminoandrostan-17

to be approximately

high for a successful

separation

in a non-specific

of magnitude

features. 1

10’ by affinity

manner,

lower than TBG,

is to be expected.

of all of the above factors

the separation

of TBG from plasma.

pure material

to allow the properties

The more pragmatic

for plasma testosterone

were

to have all of the required

its constant is about four orders

aspect

prompted

The objective

the undertaking

of

was to obtain sufficient

of this protein to be studied in some of using the protein

in displacement

assays ’

was also considered. MATERIALS

Labelled

since testo-

work on TBG ’ showed

features

substituent.

Albumin binds testosterone

Considerations

detail.

then arose

that ring A could be altered within limits without seriously

D containing

however,

steroid

The published

does not have an amino group.

quite clearly

ring

of finding an appropriate

Testosterone

:

AND METHODS

1, 2-H3-Testosterone

England Nuclear Corp. and had a specific was checked by thin layer chromatography.

activity

was purchased of 50.0

Ci/mM.

from Purity

New

STEROIDS

Sept. 1969

265

Commercially available agarose (Sepharose 4B, Pharmacia Agarose: Co.) was used without any prior treatment. 3 p-Aminoandrostan-17 p-01: This steroid was purchased from Ikapharm co. , Ramat Gan, Israel, and used without further purification. The m. p. , infrared spectrum and nuclear magnetic resonance spectrum were all consistent with the above formula. Pooled plasma was obtained through the courtesy of the Kaplan Plasma: Hospital, Rehovot, Israel. The sample contained contributions from both male and female donors and was stored under sterile conditions at 4’C. Assay for Binding Activity: The procedure followed was adapted from the results of Vermeulen and Verdonck. ’ Briefly, this involved incubation of 0.5 ml of plasma with about 100,000 dpm H3-testosterone. The bound material was separated from unbound by gel filtration chromatography on Sephadex G-25 (1 x 25 cm column). The elution patterns were exactly as those reported. All of the proteins appeared in the lo-20 mlvolume of effluent; the unbound testosterone came out in a broad peak centered at about 50 ml. Aliquots (0.2 ml) of the protein containing fraction were counted in a mixture of toluene-ethanol (10:3) containing 4 g PPO and 50 mg POPOP per liter. A Packard Tri Carb instrument was used and efficiencies were determined by the internal standard method. Preparation of Steroid-Agarose Complex: The method of Porath 4 was used exactly as described in the literature except that a steroid was used for the amino component. About 30 mg of steroid was reacted with 30 g of “activated” Sepharose and approximately 90% of the steroid was covalently bound. About 2-3 mg of steroid could be found in the filtrate from the reaction mixture and only traces of steroid could be extracted by leaching of a polymer sample with a CHC13-ethanol mixture. Separation of T. B. G. : A bed of the steroid-sepharose complex 1 cm in diameter x 2 cm high was prepared. 50 ml of plasma were passed through at room temperature at the rate of about 1.0 ml/min. The column was then washed with 0.15 M phosphate buffer (PH 7.4) until only traces of protein were being eluted. The displacing solution (1.0 M guanidine. HCl) was then passed through followed by several volumes of buffer (0.15 M phosphate, pH 7.4) to insure complete elution. The eluent was concentrated using pressure filtration through a gel membrane (Diaflo Corp. ). A “50,000 cut-off” membrane (XM - 50) was used after a control run indicated that no binding activity is lost by this procedure. RESULTS

AND DISCUSSION

The removal of TBG from plasma by affirfity chromatography is summarized in the Table below.

The first column (dpm) represents the

2G6

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STEROIDS total radioactivity

in the bound fraction

and is therefore

amount of TBG present.

The total protein

estimated

the optical

second

by determining

The ratio dpm/O.

column.

the solution with respect

content in the bound fraction

D. (r)

is a measure

Chromatography

decrease

1 (about 50 ml) contains

0. D.

(dpm)

n8epharosem demonstrating

r (dpm/O. D. )

(280 mp)

5,500 1,300 23,400 159,000 5,600 4,200

2. 8 2.6 0.080 0.015 2.4 0.63

the bulk of the plasma

An aliquot was assayed

could not be observed

after it was passed

for binding activity by untreated

when plasma was passed

that the steroid-agarose

and showed a

plasma

complex

was interacting

The question now was whether the TBG had been somehow

or whether

it could be retrieved

No significant

negligible.

(Fraction

fraction

agent was needed to remove was eluted by passing

was immediately Fraction

three times

in an effort

for binding activity. in binding activity

binding activity

density

could be detected

of the high binding constant a fairly

the TBG from the column.

A third

1.0 ml of 1 M guanidine. HCl at pH 2.1. 6 This

diluted by 10 ml of buffer

3 was concentrated

(50 ml) until the optical

2).

It was then apparent that because powerful

denatured

from the column intact.

The column was then washed with buffer

in these washings

(see Table).

over untreated

with TBG.

of the eluent became

of

of Plasma

of about 75’4 from the value exhibited

This effect

of enrichment

15,500 3,380 1,870 2,380 13,500 2,600

Untreated plasma Fraction 1 Fraction 3 Fraction 3 ’ “Sepharose” control “Diaflo” control

through the column.

was

density at 280 rnp and is shown in the

Bound Radioactivity

Fraction

of the

to TBG.

Affinity

Sample

a measure

(PH 7.4) to minimize

by ultrafiltration,

to remove

rediluted

all of the guanidine.

As can be seen from the Table, had been achieved.

denaturation.

and reconcentrated It was then assayed

about a 4 fold enrichment

267

STEROIDS

Sept. 1969

The bound fraction (10 ml) from the assay of Fraction 3 was then concentrated

to

1.0 ml by ultrafiltration

chromatography was assayed

had removed

since it was expected

all traces

of guanidine.

and indeed the binding activity

Examination

of the total radioactivity

the yield of TBG by this procedure that the guanidine does not efficiently is that considerable

protein

The concentrate

had increased in Fractions

is quite small remove

(Fraction

3 ‘)

more than 6 fold. 3 and 3’ indicates

(0.4’4).

One possibility

the adhering

is being inreversibly

that Sephadex

protein.

denatured

that is

Another

in the process.

It

3

is interesting difficulties

to note that Vonderhaar

with their receptor

The above experiments this protein

from plasma

and Mueller

have experienced

similar

proteins. with TBG demonstrate

by affinity

the possibility

chromatography.

will,

of course,

have to be improved

before

large

samples.

It is hoped that future efforts

The efficiency

of separating of the process

it can be used for the preparation on this problem

will resolve

of

this

difficulty.

ACKNOWLEDGEMENTS The author would like to express for the hospitality

extended

are also due to numerous in conducting preparation

this research.

Cancer

Society

to Professor

E. Katchalski

during the tenure of his stay in Rehovot. other members In particular

of the agarose-steroid

This project

gratitude

complex

was made possible

(Grant No. P-498).

of the Institute for advice the help of Dr. is greatly

with the financial

Thanks and help

S. Blumberg

in the

appreciated. assistance

of the American

268

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STEROIDS

REFERENCES

Steroids, 2,

609 (1968) and references,

1.

Vermeulen, A., Verdonck, cited therein.

2(a)

Baker, B. R. “Design of 4ctive-Site-Directed Irreversible Inhibitors “, Chap. 13, John Wiley and Sons (1967).

(b)

L.,

Enzyme-

Silman, I. H., Katchalski, E., Ann. Rev. Biochem. v 35_, 873 (1966).

3.

Vonderhaar, B., Muella, G. C., Biochim. Biophgs. Acta,

176, 626 (1969).

4.

Porath, J., Axen, R.,

5.

Horton, R. , Kato, T. , Sherins, R. , Steroids, 10, 245 (1967).

6.

Cuatrecasas, 235 (1968).

Ernback, S., Nature, 215, 1491 (1967).

P. , Wilchek, M. , Biochem. Biophys. Res. Comm.

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