164
GENERAL METHODOLOGY
[13]
This inhibition of aspartate aminotransferase by the inhibitor is of interest because it points up the uncontrollable aspects of these inhibition studies. A priori, one would not expect propargylglycine to be an inhibitor of this enzyme because it should not be able to activate the acetylene. Fortuitously, a basic amino acid is adjacent to the relevant C--B bond of the inhibitor and can, in fact, break it with the resulting activation of the acetylene. Conclusion
Enzymes capable of generating a earbanion or a carbanion-like intermediate adjacent to an acetylene are susceptible to irreversible inhibition by these acetylenic reagents. Flavin-linked and pyridoxal-linked enzymes and isomerases have proved to be good candidates for inhibition by the acetylenic inhibitors. This is the case because these enzymes are generally involved in C - - H bond abstraction with concomitant carbanion or carbanion-like intermediate formation. However, the critical factor is the formation of a free valance adjacent to the acetylenic unit. An enzyme that could do so is a candidate for inhibition by these inhibitors. Thymidylate synthethase, for example, should be irreversibly inhibited by 5ethynouracil; the free valance would be generated adjacent to the acetylene by means of an enzyme-catalyzed nucleophilic addition to the uracil. TM ~ H . Sommer a n d D. Senti,
Biochem. Biophys. Res. Commun. 57, 689 (1974).
[13] O r g a n i c I s o t h i o c y a n a t e s as A f f i n i t y L a b e l s By ROBERT G . LANGI)ON
Affinity labels may be regarded as being comprised of two parts: a portion, A, which associates specifically with a protein active site and a second reactive portion, C, which reacts to form a covalent linkage between a reactive group in the protein and the affinity probe itself. A -- C -~ protein --* A -- C - protein
Ideally, the C group should not interfere with interaction between the A group and its binding site. It should react with some nearby protein functional group much more readily than it interacts with solvent, and it should form a stable product. Finally, it should be easily synthesized from readily available precursors. In several ways, organic isothiocyanates satisfy these criteria rather well. The isothiocyanate group itself has no net charge and is not excessively bulky, so that it is not subject to electrostatic repulsion, and it is
[13]
165
ORGANIC ISOTI-IIOCYANATES
sufficiently small that it may avoid steric problems. Isothiocyanates react readily with amino groups in proteins to form disubstituted thioureas, which are stable under physiological conditions. On the other hand, reactivity with water is so slight that steam-volatile isothiocyanates may be purified by steam distillation. Finally, isothiocyanates are rather easily prepared by several methods. The preparation, properties, and reactions of isothiocyanates have been the subject of valuable, comprehensive reviews, which should be consulted for details. 1,2 Synthesis of Isothiocyanates Preparative methods that are available include the following: 1. From alkyl halides and metal thiocyanates: R - Cl + NH~SCN ~ R - NCS ( + RSCN) + NH4C1
2. From salts of dithiocarbambic acid derivatives: S II
(a) R--NHCSNH4 -F Pb(NOs)2---* RNCS + NH~NOs -F HNOs + PbS S
i[
(b) R--NHCSNH4 -F COC12 --* RNCS + NH4C1 -{- HC1 -t- COS S
ti
(c) R--NHCSNH4 -b 4NaOC1 + NaOH RNCS -F 3NaC1 + NH4C1 + Na~SO4 -F H20
3. From disubstituted thioureas: H
S
H
I
il
i
R--N--C--N--R
HCl, h~,,t
) RNCS + R--NHsC1
4. From primary amines and thiocarbonyl diimidazole3: N
:k
S
ti
/~
N
S:
~i
/:t
N
RHN--C--N
\_
~N
\__
+ H
R--NCS + H N \ C N 1 M. BSgemann, S. Petersen, O. E. Schultz, and H. $511, in Houben-Weyl "Methoden der Organischen Chemie" (E. Miiller, ed.), Vol. IX, pp. 773-913. Thieme, Stuttgart, 1955. 2S. J. Assony, in "Organic Sulfur Compounds" (N. Kharasch, ed.), Vol. I, pp. 326338. Pergamon, London, 1961. s H. A. Staab and G. Walther, Justus Liebigs Ann. Chem. 657, 98 (1962).
166
GENERAL METHODOLOGY
[13]
5. From primary amines and thiophosgene: S
II
R" NH2 + CSC12--* [R--NH--C--C1] + HC1 R--NCS + HC1
Method 5, the reaction of thiophosgene with primary amines, is probably the most generally useful, since primary amines are frequently the most readily available of the starting materials, either by purchase or by synthesis, and thiophosgene is commercially available. Furthermore, the reaction may be carried out either in organic solvents or in aqueous solutions of water-soluble amines, because both thiophosgene and isothiocyanares react only slowly with cold water near neutrality; thiophosgene is sufficiently soluble in water so that the reaction proceeds well. The amine may be used as the free base or, if it is sufficiently basic, in its protonated form; the latter is usually preferable because it prevents undesired reaction of the synthesized isothiocyanate with its precursor amine and results in higher overall yields. The major disadvantage of this method is that thiophosgene, a liquid at room temperature (b.p. 73.5°), is very toxic and must be used in an efficient fume hood with precautions adequate to prevent skin contact or inhalation of its vapor. For storage, small screwcapped bottles of thiophosgene are placed in a desiccator, which is kept at --20°; prior to use, the desiccator is allowed to come to room temperature in the fume hood. The following description of the preparation of D-glucosyl isothiocyanate 4 is illustrative of the method. To 71.2 mg (400 ~moles) of glucosylamine dissolved in 4.0 ml of 0.4 M NaHC03 (1600 ~moles) at room temperature in a glass-stoppered tube are added 50 ~l (657 ~moles) of CSC12 with a microliter syringe to effect the transfer. The mixture, stirred rapidly with a magnetic stirrer, is allowed to react for 12 rain. At the end of this period, evolution of CO~ should have stopped or become quite slow, and droplets of orange-colored thiophosgene should have disappeared. To the mixture are added 15 ml of dichloromethane. The tube is stoppered, shaken, and centrifuged at low speed. The subnatant dichloromethane solution containing residual dissolved thiophosgene is removed with a syringe and transferred into a beaker containing ammonium hydroxide (hood!). The upper aqueous phase is extracted again with a second 15-ml portion of dichloromethane, and, after centrifugation, the upper aqueous phase is transferred to a fresh tube with care to avoid contamination with 4 R. D. Taverna and R. G. Langdon, Biochem. Biophys. Res. Commun. 54, 593 (1973).
[13]
ORG&NIC ISOTHIOCYANATES
167
dichloromethane. The aqueous glucosyl isothiocyanate solution is placed in an ice bath and used within 30 min. Reactions of Isothiocyanates with Protein Functional Groups
The reactions of isothiocyanates with proteins have been extensively studied, particularly in connection with the Edman procedure. ~-7 The major initial reaction that occurs under mild conditions at neutral or slightly alkaline pH is the formation of thiourea derivatives with amino terminal residues and with c-amino groups of lysyl residues. R-NCS + NH2 - protein--* R - - N - - C - - N - protein H
Although further modification, e.g., substituted thiohydantoin formation, may result under other circumstances, thiourea derivatives are ordinarily quite stable near neutrality. Isothiocyanate derivatives of stilbene sulfonates have been utilized with marked success as affinity labels for an anion transport protein of the erythrocyte membrane by Rothstein and his associates, s,9 and glucosyl isothiocyanate has been employed to label a glucose transport protein of the human erythrocyte membrane. 4 Both proteins fall in the 90,000-100,000 dalton range (band 3 in the terminology of Fairbanks et a/.l°). In both cases, transport has been reconstituted using phospholipid vesicles and a band 3 preparation T M after affinity labeling with the appropriate isothiocyanate had provided preliminary evidence that this protein class might be involved in transport. Because affinity labels may differ in the avidity with which they are bound, proximity of their binding site to an amino group on the protein, and in other respects, no specific directions can be given for interaction of isothiocyanate affinity labels with proteins which would be universally applicable. In general, a substrate analog containing an isothiocyanate reactive group is incubated at different concentrations for varying periods of time 5 p. Edman, Acta Chem. Scand. 10, 761 (1956). W. A. Schroeder, this series, Vol. 25, p. 298 (1972). H. Fraenkel-Conrat, J. I. Harris, and A. L. Levy, Methods Biochem. Anal. 2, 383 (1955). 8Z. I. Cabantchik and A. l%thstein, J. Membr. Biol. 10, 311 (1972). A. Rothstein, Z. I. Cabantchik, and P. Knauf, Fed. Proc., Fed. Am. Soc. Exp. Biol. 35, 3 (1976). t°G. Fairbanks, T. L. Steck, and D. F. H. Wallach, Biochemistry 10, 2606 (1971). 11M. Kasahara and P. C. Hinkle, Proc. Natl. Acad. Sci. U,S.A. 73, 396 (1976).
168
GENERAL METHODOLOGY
[14]
with the protein or membrane of interest in a neutral or alkaline buffer free of ammonia, amines, or mercaptans, and conditions are chosen such that approximately 50% inhibition of function is observed. Experiments are then conducted to ascertain whether a normal substrate protects competitively against the inhibitory action of the presumed affinity label. Finally, the isothiocyanate-containing affinity label may be prepared in radioactive form, and its specific interaction with a particular protein among a mixture of proteins may be examined; again, demonstration of competitive inhibition of reaction by a normal substrate or by a potent reversible competitive inhibitor is a valuable or essential criterion for specificity of interaction between the affinity label and the protein of interest. As a specific example of methodolgy, reaction of D-glucosyl isothiocyanate with the erythrocyte membrane may be cited. 4 In preliminary experiments 50% suspensions of washed human erythrocytcs in Krebs-Ringer phosphate buffer at pH 7.4 were incubated at 37 ° with 0-100 mM D-glucosyl isothiocyanate for periods of 0-60 min. After incubation, the cells were washed twice with 20 volumes of ice-cold buffer and the rates of influx of 30 mM [14C]glucose were measured. It was found that prior treatment of the cells with 20 mM D-glucosyl isothiocyanate at 37 ° for 5 min resulted in 50% inhibition of the transport rate. The presence of 100 mM maltose or D-glucose during exposure to the isothiocyanate protected against inhibition whereas L-glucose was without effect. When 20 mM D-[14C]glucosyl isothiocyanate was incubated with intact erythrocytes at 37 ° for 5 min, incorporation into a number of membrane proteins as well as into hemoglobin occurred. The simultaneous presence of 100 mM D-glucose inhibited incorporation of the labeled material, particularly into band 3, while L-glucose had no effect. Limitations. Isothiocyanates react rather rapidly with exposed amino groups, whether or not they are attached to affinity groups. Therefore, nonspecific reaction can be expected to occur, and the investigator must demonstrate that the effects he observes with isothiocyanate affinity labels are indeed specific effects rather than the result of nonspecific interaction.
[14] P h o t o - C r o s s - L i n k i n g o f P r o t e i n - N u c l e i c Acid Complexes
By PAUL R. SCHIMMEL and GERALD P. BUDZIK In recent years the rapid expansion of biological research has resulted in an increasing interest in macromolecular complexes. This interest has
GENERAL METHODOLOGY
[13]
This inhibition of aspartate aminotransferase by the inhibitor is of interest because it points up the uncontrollable aspects of these inhibition studies. A priori, one would not expect propargylglycine to be an inhibitor of this enzyme because it should not be able to activate the acetylene. Fortuitously, a basic amino acid is adjacent to the relevant C--B bond of the inhibitor and can, in fact, break it with the resulting activation of the acetylene. Conclusion
Enzymes capable of generating a earbanion or a carbanion-like intermediate adjacent to an acetylene are susceptible to irreversible inhibition by these acetylenic reagents. Flavin-linked and pyridoxal-linked enzymes and isomerases have proved to be good candidates for inhibition by the acetylenic inhibitors. This is the case because these enzymes are generally involved in C - - H bond abstraction with concomitant carbanion or carbanion-like intermediate formation. However, the critical factor is the formation of a free valance adjacent to the acetylenic unit. An enzyme that could do so is a candidate for inhibition by these inhibitors. Thymidylate synthethase, for example, should be irreversibly inhibited by 5ethynouracil; the free valance would be generated adjacent to the acetylene by means of an enzyme-catalyzed nucleophilic addition to the uracil. TM ~ H . Sommer a n d D. Senti,
Biochem. Biophys. Res. Commun. 57, 689 (1974).
[13] O r g a n i c I s o t h i o c y a n a t e s as A f f i n i t y L a b e l s By ROBERT G . LANGI)ON
Affinity labels may be regarded as being comprised of two parts: a portion, A, which associates specifically with a protein active site and a second reactive portion, C, which reacts to form a covalent linkage between a reactive group in the protein and the affinity probe itself. A -- C -~ protein --* A -- C - protein
Ideally, the C group should not interfere with interaction between the A group and its binding site. It should react with some nearby protein functional group much more readily than it interacts with solvent, and it should form a stable product. Finally, it should be easily synthesized from readily available precursors. In several ways, organic isothiocyanates satisfy these criteria rather well. The isothiocyanate group itself has no net charge and is not excessively bulky, so that it is not subject to electrostatic repulsion, and it is
[13]
165
ORGANIC ISOTI-IIOCYANATES
sufficiently small that it may avoid steric problems. Isothiocyanates react readily with amino groups in proteins to form disubstituted thioureas, which are stable under physiological conditions. On the other hand, reactivity with water is so slight that steam-volatile isothiocyanates may be purified by steam distillation. Finally, isothiocyanates are rather easily prepared by several methods. The preparation, properties, and reactions of isothiocyanates have been the subject of valuable, comprehensive reviews, which should be consulted for details. 1,2 Synthesis of Isothiocyanates Preparative methods that are available include the following: 1. From alkyl halides and metal thiocyanates: R - Cl + NH~SCN ~ R - NCS ( + RSCN) + NH4C1
2. From salts of dithiocarbambic acid derivatives: S II
(a) R--NHCSNH4 -F Pb(NOs)2---* RNCS + NH~NOs -F HNOs + PbS S
i[
(b) R--NHCSNH4 -F COC12 --* RNCS + NH4C1 -{- HC1 -t- COS S
ti
(c) R--NHCSNH4 -b 4NaOC1 + NaOH RNCS -F 3NaC1 + NH4C1 + Na~SO4 -F H20
3. From disubstituted thioureas: H
S
H
I
il
i
R--N--C--N--R
HCl, h~,,t
) RNCS + R--NHsC1
4. From primary amines and thiocarbonyl diimidazole3: N
:k
S
ti
/~
N
S:
~i
/:t
N
RHN--C--N
\_
~N
\__
+ H
R--NCS + H N \ C N 1 M. BSgemann, S. Petersen, O. E. Schultz, and H. $511, in Houben-Weyl "Methoden der Organischen Chemie" (E. Miiller, ed.), Vol. IX, pp. 773-913. Thieme, Stuttgart, 1955. 2S. J. Assony, in "Organic Sulfur Compounds" (N. Kharasch, ed.), Vol. I, pp. 326338. Pergamon, London, 1961. s H. A. Staab and G. Walther, Justus Liebigs Ann. Chem. 657, 98 (1962).
166
GENERAL METHODOLOGY
[13]
5. From primary amines and thiophosgene: S
II
R" NH2 + CSC12--* [R--NH--C--C1] + HC1 R--NCS + HC1
Method 5, the reaction of thiophosgene with primary amines, is probably the most generally useful, since primary amines are frequently the most readily available of the starting materials, either by purchase or by synthesis, and thiophosgene is commercially available. Furthermore, the reaction may be carried out either in organic solvents or in aqueous solutions of water-soluble amines, because both thiophosgene and isothiocyanares react only slowly with cold water near neutrality; thiophosgene is sufficiently soluble in water so that the reaction proceeds well. The amine may be used as the free base or, if it is sufficiently basic, in its protonated form; the latter is usually preferable because it prevents undesired reaction of the synthesized isothiocyanate with its precursor amine and results in higher overall yields. The major disadvantage of this method is that thiophosgene, a liquid at room temperature (b.p. 73.5°), is very toxic and must be used in an efficient fume hood with precautions adequate to prevent skin contact or inhalation of its vapor. For storage, small screwcapped bottles of thiophosgene are placed in a desiccator, which is kept at --20°; prior to use, the desiccator is allowed to come to room temperature in the fume hood. The following description of the preparation of D-glucosyl isothiocyanate 4 is illustrative of the method. To 71.2 mg (400 ~moles) of glucosylamine dissolved in 4.0 ml of 0.4 M NaHC03 (1600 ~moles) at room temperature in a glass-stoppered tube are added 50 ~l (657 ~moles) of CSC12 with a microliter syringe to effect the transfer. The mixture, stirred rapidly with a magnetic stirrer, is allowed to react for 12 rain. At the end of this period, evolution of CO~ should have stopped or become quite slow, and droplets of orange-colored thiophosgene should have disappeared. To the mixture are added 15 ml of dichloromethane. The tube is stoppered, shaken, and centrifuged at low speed. The subnatant dichloromethane solution containing residual dissolved thiophosgene is removed with a syringe and transferred into a beaker containing ammonium hydroxide (hood!). The upper aqueous phase is extracted again with a second 15-ml portion of dichloromethane, and, after centrifugation, the upper aqueous phase is transferred to a fresh tube with care to avoid contamination with 4 R. D. Taverna and R. G. Langdon, Biochem. Biophys. Res. Commun. 54, 593 (1973).
[13]
ORG&NIC ISOTHIOCYANATES
167
dichloromethane. The aqueous glucosyl isothiocyanate solution is placed in an ice bath and used within 30 min. Reactions of Isothiocyanates with Protein Functional Groups
The reactions of isothiocyanates with proteins have been extensively studied, particularly in connection with the Edman procedure. ~-7 The major initial reaction that occurs under mild conditions at neutral or slightly alkaline pH is the formation of thiourea derivatives with amino terminal residues and with c-amino groups of lysyl residues. R-NCS + NH2 - protein--* R - - N - - C - - N - protein H
Although further modification, e.g., substituted thiohydantoin formation, may result under other circumstances, thiourea derivatives are ordinarily quite stable near neutrality. Isothiocyanate derivatives of stilbene sulfonates have been utilized with marked success as affinity labels for an anion transport protein of the erythrocyte membrane by Rothstein and his associates, s,9 and glucosyl isothiocyanate has been employed to label a glucose transport protein of the human erythrocyte membrane. 4 Both proteins fall in the 90,000-100,000 dalton range (band 3 in the terminology of Fairbanks et a/.l°). In both cases, transport has been reconstituted using phospholipid vesicles and a band 3 preparation T M after affinity labeling with the appropriate isothiocyanate had provided preliminary evidence that this protein class might be involved in transport. Because affinity labels may differ in the avidity with which they are bound, proximity of their binding site to an amino group on the protein, and in other respects, no specific directions can be given for interaction of isothiocyanate affinity labels with proteins which would be universally applicable. In general, a substrate analog containing an isothiocyanate reactive group is incubated at different concentrations for varying periods of time 5 p. Edman, Acta Chem. Scand. 10, 761 (1956). W. A. Schroeder, this series, Vol. 25, p. 298 (1972). H. Fraenkel-Conrat, J. I. Harris, and A. L. Levy, Methods Biochem. Anal. 2, 383 (1955). 8Z. I. Cabantchik and A. l%thstein, J. Membr. Biol. 10, 311 (1972). A. Rothstein, Z. I. Cabantchik, and P. Knauf, Fed. Proc., Fed. Am. Soc. Exp. Biol. 35, 3 (1976). t°G. Fairbanks, T. L. Steck, and D. F. H. Wallach, Biochemistry 10, 2606 (1971). 11M. Kasahara and P. C. Hinkle, Proc. Natl. Acad. Sci. U,S.A. 73, 396 (1976).
168
GENERAL METHODOLOGY
[14]
with the protein or membrane of interest in a neutral or alkaline buffer free of ammonia, amines, or mercaptans, and conditions are chosen such that approximately 50% inhibition of function is observed. Experiments are then conducted to ascertain whether a normal substrate protects competitively against the inhibitory action of the presumed affinity label. Finally, the isothiocyanate-containing affinity label may be prepared in radioactive form, and its specific interaction with a particular protein among a mixture of proteins may be examined; again, demonstration of competitive inhibition of reaction by a normal substrate or by a potent reversible competitive inhibitor is a valuable or essential criterion for specificity of interaction between the affinity label and the protein of interest. As a specific example of methodolgy, reaction of D-glucosyl isothiocyanate with the erythrocyte membrane may be cited. 4 In preliminary experiments 50% suspensions of washed human erythrocytcs in Krebs-Ringer phosphate buffer at pH 7.4 were incubated at 37 ° with 0-100 mM D-glucosyl isothiocyanate for periods of 0-60 min. After incubation, the cells were washed twice with 20 volumes of ice-cold buffer and the rates of influx of 30 mM [14C]glucose were measured. It was found that prior treatment of the cells with 20 mM D-glucosyl isothiocyanate at 37 ° for 5 min resulted in 50% inhibition of the transport rate. The presence of 100 mM maltose or D-glucose during exposure to the isothiocyanate protected against inhibition whereas L-glucose was without effect. When 20 mM D-[14C]glucosyl isothiocyanate was incubated with intact erythrocytes at 37 ° for 5 min, incorporation into a number of membrane proteins as well as into hemoglobin occurred. The simultaneous presence of 100 mM D-glucose inhibited incorporation of the labeled material, particularly into band 3, while L-glucose had no effect. Limitations. Isothiocyanates react rather rapidly with exposed amino groups, whether or not they are attached to affinity groups. Therefore, nonspecific reaction can be expected to occur, and the investigator must demonstrate that the effects he observes with isothiocyanate affinity labels are indeed specific effects rather than the result of nonspecific interaction.
[14] P h o t o - C r o s s - L i n k i n g o f P r o t e i n - N u c l e i c Acid Complexes
By PAUL R. SCHIMMEL and GERALD P. BUDZIK In recent years the rapid expansion of biological research has resulted in an increasing interest in macromolecular complexes. This interest has
