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Preparation of single crystals of transferrin
.4RC’HIVES
OF
BIOCHEMISTRY
AND
121, 717-719
BIOPHYSICS
Preparation
of Single
ADELA
J. LEIBI\‘IAN
IBM Watson Laboratory,
Columbia
l
(1967)
Crystals
PHILIP
AND University,
1967;
of Transferrin
New
accepted
April
AISEN I’ork,
Xew
York
10025
11, 19G7
A convenient and reproducible method for the crystallization of transferrin utilizing a collodion membrane ultrafiltration apparatus is presented. The method yields crystals that may be frozen for low-temperature studies without significant loss of crystallinity. Some of the physical properties of the crystallized transferrin are presented.
The current int#erest in macromolecular structure has created a need for simplified methods of preparing single crystals of prot,eins suit,able for analysis by X-ray diffraction,
electron
k>aramagnetic
resonance
matographic step may be repeated, bnt, this is 1~. necessary if the prodllct shows a ratio of absorhanre at, 470 mp to adsorbance at 410 111p exceeding 1.3. After overnight dialysis against running tap lvater the preparation was concentrated by ldtrafiltration lentil the absorbancy at 470 111~ was between 6.0 alld 8.0. The concentrated solution was dialyzed overnight against 0.01 Y sodium acetate bluffer, pII 6.0, alld placed inside the collodioll membrane bag of the Schleicher and Schuell Idtrafiltration apparatlls. The sllrt.ioll vessel is halffilled with cold 157; ethanol in 0.01 M sodinrtl acetate butier, pI1 6.0, evacrtated, alld allowed to stand with occasional stirring, in the refrigerator or cold room. After several hours tiny crystals became visible; when a heavy deposit was collected the preparation was centrifuged in the cold. The mixed amorphous-crystalline material was redissolved in a minimlml volrnne of distilled water and rent rifuged to remove any insoluble material. The preparation was again placed in t hc nlenbrane bag of the Idtrafiltration apparattls, 1 he SI~Clion vessel collt:tiIling this time Syc ethanol in 0.005 M sodirnn acetate, pl* 6.0. After evaclla(ion small well-formed crystals appeared on standing in the cold, llsrlally within 2 hours. The preparation was then warmed to room temperatt7re, trailsferred to a small test tllbe, and left luldistrn+ed at 0°C. The crystals continlled togrow, and reached a size of several millimeters within a few da>3 (Fig. 1) ; individllal sitlglr crystals \verr readily barvested.
spec-
troscopy, and related techniques. Recently we have improved upon a method for cryst,allizing kansferrin which makes possiblethe couvenient
preparation
of large
single
crys-
tals of transferrin of great’ Llurity. F’eatures of this method may also he useful in the k)reparation
of
large
single
cryst,als
of other
proteins. MATERIALS
ANI>
METHOlS
The 60-75’;; ammonilm~ sulfate fraction from a liter of pooled pig serunl (oht,ained fresh from a local slaughter horlse) was prepared by the method of Holmberg and Lanrell (1). This precipitate was dissolved in a minimum volume of water and dialyzed overnight against rrurning tap water. After clarification of the dialyzate by centrifngation, the pH was adjnsted to 9.0 with 5% NH&H solution, and half a volnnle of a 0.7$0 aqueous solution of Rivanol (Wirrt hrop Laboratories) was added dropwise with continllous stirring. The tenacious precipitate which resulted was removed by filtration throllgh a Biichner ftmnel and Whatman No. 1 paper, with the aid of sllction. The Rivanol was then adsorbed on Korit, A (2), and the crude salmall-pink solution of transferrill \vas dialyzed against 0.05 M Tris bIttier, pIl 8.0, preparatory to loading onto a 5 X 25.~111 collnnl~ of l)f’,AE-Sephadex A-50 equilibrated with the same buffer. Ellltion was accomplished \vith a gradient, of Tris cclncentration ranging from 0.05 to 0.2 11. The chro-
IL Properties
AL TS
oj crystalline
ti~amfeuiu.
The
properties of the cqstalline transfcrrin are given in Table I. The values for the (astine717
71s
LEIBRIA?;
tion coefficient of the apoprotein and t’he iron-binding capacity lead to a minimum molecular weight of 40,500, or 81,000 if 2 Fe3+ ions are bound to each molecule of protein. Although the extinction coefficients at 280 and 470 mp are about 10% higher than those reported earlier (3)) the molecular weight calculated from them is consistent with that found by sedimentation equilibrium,] and we believe t#he higher values reflect the greater degree of purity of the present preparation. It should be noted that the molarity of a protein solution calculated from the earlier vaIues for extinction coefficient and molecular weight are substant.ially the same as those which would be obtained with the present values. Preliminary data’ indicate that the molecular weight of transferrin is unchanged in 6 M guanidine hydrochloride with 0.1 M /3mercaptoet,hanol, a solvent medium in which proteins are normally dissociated to their 1 The studies in the ultracentrifuge were carried out by A.C. Cox and C. Tanford, at Duke University. We are grateful for permission to publish the results in this paper.
AND
AISEX TABLE ~‘ROPERTIES
OF CRYSTALLINE
Property
Transferrin
Molecular weight (from ironbinding) hlolecrllar weight (sedimentation equilibrium)a Sedimentation velocity,” s&,~,,
TRANSFERRIK Apotransferrin
81,000
82,OOOb
5.2
E:%i,
$7 1% 2 4mmp Absorbance at mr/pg Fe3+/ml A 47amp . A41 amp
I
470
14.1 0.600 0.0454
f
0.1s 11.4
-
1.45
a See footnote 1 in text. b The methods of Yphantis (5) and LaBar were used. A 2) of 0.725 ml/gm was utilized calculation (7).
(6) fol
constituent polypeptide chains. If this result is confirmed by studies now in progress, it would indicate that transferrin consists of a single polypeptide chain even though it has two apparently identical binding sites for iron. DISCUSSIOK
/+-Imm--‘A FIG. 1. A single crystal as described in Materials
of transferrin Melhods.
and
prepared
The major advantage of the present method over those in which cryst’allization is induced by the stepwise addition of alcohol (4), or by allowing alcohol to dialyze into a protein solution, is that t,he concentrabion of protein is maintained or increased as the desired concentration of alcohol is attained. Furthermore, the optimum concentration of alcohol, when crystallizabion is first evident, is readily determined, so that trial-and-error additions are avoided (4). Transferrin crystals prepared by this method may be frozen by immersion in liquid nitrogen without significant loss of crystalline charact’er. Presumably t,he alcohol serves to disrupt the structure of the water of crystallization sufficiently so t,hat it freezes as a glass rather than in an ice lattice, and thus keeps the regions of mosaicit’y or disorder in the prot,ein cry&al to a minimum. This may be analogous to the effects of high concentrations of perchlorate on the structure of solvent mt#aerduring the freezing of
PREPARATION
OF
TRANSFERRIK
cupric peptides, as discussed by Falk et al. (8). Thus the single crystals we have grown have been highly satisfactory for electron paramagnetic resonance studies at the temperature of liquid helium (9). REFERENCES 1. LIIURELL, C.B., AND INGELMAN,B., ACta Chem. Stand. 1, 770 (1947). 2. BOETTCHER. E. W.. KISTLER. P.. AND T\TITSCHM~NX, II., N&re 181, 49b (1958). 3. AISEN, P., LIEBMAN, A., AND REICH, H. A., J. Biol. Chenl. 241, 1666 (1966).
4. LAURELL,
CRYSTALS
719
C. B., :Icfa C’hem. Stand. 7, 1407 (1953). 5. YPHANTIS, II. A., BiocAe~nistq/ 3, 297 (1964). 6. LABAR, F. E., Proc. Natl. dcad. Sci. 77.8. 54, 31 (1965). 7. SCHULTZ, H. E., AXD SCHWI~K, G., C‘lin. C’hcm. Acta 4, 15 (1959); cited by F. W. Putnam in “The Proteins” (H. Neurath, ed.), Vol. 3, p. 175. Academic Press, New York (1965). 8. FALK, K.E., 1-I. C.,JANSSON, T., &~ALMSTROX, B. G., AND VANNGARD, T., to be published. 9. BLUMBERT, W., AND AISEN, P., unpublished observations.
OF
BIOCHEMISTRY
AND
121, 717-719
BIOPHYSICS
Preparation
of Single
ADELA
J. LEIBI\‘IAN
IBM Watson Laboratory,
Columbia
l
(1967)
Crystals
PHILIP
AND University,
1967;
of Transferrin
New
accepted
April
AISEN I’ork,
Xew
York
10025
11, 19G7
A convenient and reproducible method for the crystallization of transferrin utilizing a collodion membrane ultrafiltration apparatus is presented. The method yields crystals that may be frozen for low-temperature studies without significant loss of crystallinity. Some of the physical properties of the crystallized transferrin are presented.
The current int#erest in macromolecular structure has created a need for simplified methods of preparing single crystals of prot,eins suit,able for analysis by X-ray diffraction,
electron
k>aramagnetic
resonance
matographic step may be repeated, bnt, this is 1~. necessary if the prodllct shows a ratio of absorhanre at, 470 mp to adsorbance at 410 111p exceeding 1.3. After overnight dialysis against running tap lvater the preparation was concentrated by ldtrafiltration lentil the absorbancy at 470 111~ was between 6.0 alld 8.0. The concentrated solution was dialyzed overnight against 0.01 Y sodium acetate bluffer, pII 6.0, alld placed inside the collodioll membrane bag of the Schleicher and Schuell Idtrafiltration apparatlls. The sllrt.ioll vessel is halffilled with cold 157; ethanol in 0.01 M sodinrtl acetate butier, pI1 6.0, evacrtated, alld allowed to stand with occasional stirring, in the refrigerator or cold room. After several hours tiny crystals became visible; when a heavy deposit was collected the preparation was centrifuged in the cold. The mixed amorphous-crystalline material was redissolved in a minimlml volrnne of distilled water and rent rifuged to remove any insoluble material. The preparation was again placed in t hc nlenbrane bag of the Idtrafiltration apparattls, 1 he SI~Clion vessel collt:tiIling this time Syc ethanol in 0.005 M sodirnn acetate, pl* 6.0. After evaclla(ion small well-formed crystals appeared on standing in the cold, llsrlally within 2 hours. The preparation was then warmed to room temperatt7re, trailsferred to a small test tllbe, and left luldistrn+ed at 0°C. The crystals continlled togrow, and reached a size of several millimeters within a few da>3 (Fig. 1) ; individllal sitlglr crystals \verr readily barvested.
spec-
troscopy, and related techniques. Recently we have improved upon a method for cryst,allizing kansferrin which makes possiblethe couvenient
preparation
of large
single
crys-
tals of transferrin of great’ Llurity. F’eatures of this method may also he useful in the k)reparation
of
large
single
cryst,als
of other
proteins. MATERIALS
ANI>
METHOlS
The 60-75’;; ammonilm~ sulfate fraction from a liter of pooled pig serunl (oht,ained fresh from a local slaughter horlse) was prepared by the method of Holmberg and Lanrell (1). This precipitate was dissolved in a minimum volume of water and dialyzed overnight against rrurning tap water. After clarification of the dialyzate by centrifngation, the pH was adjnsted to 9.0 with 5% NH&H solution, and half a volnnle of a 0.7$0 aqueous solution of Rivanol (Wirrt hrop Laboratories) was added dropwise with continllous stirring. The tenacious precipitate which resulted was removed by filtration throllgh a Biichner ftmnel and Whatman No. 1 paper, with the aid of sllction. The Rivanol was then adsorbed on Korit, A (2), and the crude salmall-pink solution of transferrill \vas dialyzed against 0.05 M Tris bIttier, pIl 8.0, preparatory to loading onto a 5 X 25.~111 collnnl~ of l)f’,AE-Sephadex A-50 equilibrated with the same buffer. Ellltion was accomplished \vith a gradient, of Tris cclncentration ranging from 0.05 to 0.2 11. The chro-
I
AL TS
oj crystalline
ti~amfeuiu.
The
properties of the cqstalline transfcrrin are given in Table I. The values for the (astine717
71s
LEIBRIA?;
tion coefficient of the apoprotein and t’he iron-binding capacity lead to a minimum molecular weight of 40,500, or 81,000 if 2 Fe3+ ions are bound to each molecule of protein. Although the extinction coefficients at 280 and 470 mp are about 10% higher than those reported earlier (3)) the molecular weight calculated from them is consistent with that found by sedimentation equilibrium,] and we believe t#he higher values reflect the greater degree of purity of the present preparation. It should be noted that the molarity of a protein solution calculated from the earlier vaIues for extinction coefficient and molecular weight are substant.ially the same as those which would be obtained with the present values. Preliminary data’ indicate that the molecular weight of transferrin is unchanged in 6 M guanidine hydrochloride with 0.1 M /3mercaptoet,hanol, a solvent medium in which proteins are normally dissociated to their 1 The studies in the ultracentrifuge were carried out by A.C. Cox and C. Tanford, at Duke University. We are grateful for permission to publish the results in this paper.
AND
AISEX TABLE ~‘ROPERTIES
OF CRYSTALLINE
Property
Transferrin
Molecular weight (from ironbinding) hlolecrllar weight (sedimentation equilibrium)a Sedimentation velocity,” s&,~,,
TRANSFERRIK Apotransferrin
81,000
82,OOOb
5.2
E:%i,
$7 1% 2 4mmp Absorbance at mr/pg Fe3+/ml A 47amp . A41 amp
I
470
14.1 0.600 0.0454
f
0.1s 11.4
-
1.45
a See footnote 1 in text. b The methods of Yphantis (5) and LaBar were used. A 2) of 0.725 ml/gm was utilized calculation (7).
(6) fol
constituent polypeptide chains. If this result is confirmed by studies now in progress, it would indicate that transferrin consists of a single polypeptide chain even though it has two apparently identical binding sites for iron. DISCUSSIOK
/+-Imm--‘A FIG. 1. A single crystal as described in Materials
of transferrin Melhods.
and
prepared
The major advantage of the present method over those in which cryst’allization is induced by the stepwise addition of alcohol (4), or by allowing alcohol to dialyze into a protein solution, is that t,he concentrabion of protein is maintained or increased as the desired concentration of alcohol is attained. Furthermore, the optimum concentration of alcohol, when crystallizabion is first evident, is readily determined, so that trial-and-error additions are avoided (4). Transferrin crystals prepared by this method may be frozen by immersion in liquid nitrogen without significant loss of crystalline charact’er. Presumably t,he alcohol serves to disrupt the structure of the water of crystallization sufficiently so t,hat it freezes as a glass rather than in an ice lattice, and thus keeps the regions of mosaicit’y or disorder in the prot,ein cry&al to a minimum. This may be analogous to the effects of high concentrations of perchlorate on the structure of solvent mt#aerduring the freezing of
PREPARATION
OF
TRANSFERRIK
cupric peptides, as discussed by Falk et al. (8). Thus the single crystals we have grown have been highly satisfactory for electron paramagnetic resonance studies at the temperature of liquid helium (9). REFERENCES 1. LIIURELL, C.B., AND INGELMAN,B., ACta Chem. Stand. 1, 770 (1947). 2. BOETTCHER. E. W.. KISTLER. P.. AND T\TITSCHM~NX, II., N&re 181, 49b (1958). 3. AISEN, P., LIEBMAN, A., AND REICH, H. A., J. Biol. Chenl. 241, 1666 (1966).
4. LAURELL,
CRYSTALS
719
C. B., :Icfa C’hem. Stand. 7, 1407 (1953). 5. YPHANTIS, II. A., BiocAe~nistq/ 3, 297 (1964). 6. LABAR, F. E., Proc. Natl. dcad. Sci. 77.8. 54, 31 (1965). 7. SCHULTZ, H. E., AXD SCHWI~K, G., C‘lin. C’hcm. Acta 4, 15 (1959); cited by F. W. Putnam in “The Proteins” (H. Neurath, ed.), Vol. 3, p. 175. Academic Press, New York (1965). 8. FALK, K.E., 1-I. C.,JANSSON, T., &~ALMSTROX, B. G., AND VANNGARD, T., to be published. 9. BLUMBERT, W., AND AISEN, P., unpublished observations.