~7]
DNA-DEPENDENT RNA POLYMERASE
[37] D N A - D e p e n d e n t
353
RNA Polymerase 1
By A. P. KIMBALL An essential lysine residue in the active center of the catalytic subunit of DNA-dependent RNA polymerase can be affinity labeled with methylthioinosinedicarboxaldehyde (MMPR-0p).2, 3 The c-amino group of the lysine forms a Schiff base with an aldehyde group of MMPR-OP, which can be converted to the stable amine by mild reduction with sodium borohydride. The [8~S]MMPR-OP can be prepared easily for radioactive affinity labeling of the enzyme.
SCH3 HOCH2
[
MMPR-OP Preparation of Methylthioinosinedicarboxaldehyde (MMPR-OP) Synthesis o] Unlabeled MMPR-OP One millimole of 9-fl-~-ribofuranosyl-6-methylthiopurine (298 mg) (methylthioinosine, MTI) is allowed to react with 1.1 mmoles of periodic acid (H~106, 251 rag) in 11 ml of distilled water in the dark for 30 min. The solution is rapidly pipetted onto a Dowex 1-formate column (10 X 30 mm), and the self-eluate and two water washes are collected in a 100-ml round-bottom flask. The solution of MMPR-0P, approximately 30 mi, is frozen in a thin film around the sides of the flask by rotation in a Dry Ice-acetone mixture, and the frozen solution is immediately lyophilized to give a white fluffy powder. A yield of 70-80% can be expected. The MMPR-OP should be stored dry at --20 °. Solutions of 'This research was supported by research grants from the Robert A. Welch Foundation (E-321) and from the NCI (CA-12327) of National Institutes of Health. 2j. Nixon, T. Spoor, J. Evans, and A. P. Kimball, Biochemistry 11, 4570 (1972). 3T. C. Spoor, F, L. Persico, J. E. Evans, and A. P. Kimball, Nature (London) 227, 57 (1970).
354
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[37]
MMPR-OP should be prepared immediately before use. Any excess solution should be discarded since the MMPR-OP slowly degrades in solution to 6-methylthiopurine with a half-life of about 4 days. The MMPR-OP is moderately difficult to dissolve in water, but heat may not be used to aid solution because this rapidly degrades the MMPR-OP.
Synthesis o] [8~S]MMPR-OP One millimole (284 mg) of 9-fl-D-ribofuranosyl-6-thiopurine (thioinosine, mercaptopurine riboside, MPR) is allowed to react with 1 mmole (32 mg) of [35S]rhombic sulfur, 20 mCi/mmole, in 25 ml of dr)" pyridine under reflux for 4 hr. 4 Care should be taken to keep the MPR from accumulating around the wall of the round-bottom flask at the solution surface if a heating mantle is used, since charring may occur. After 4 hr of reflux, 25 ml of distilled water is added to the pyridine solution, and the solution is taken to dryness under reduced pressure at 40 ° . Water, 25 ml, is added to the flask and gently heated in a water bath to dissolve the [35S]MPR. A yellow-gray film that does not dissolve is [aSS]rhombic sulfur. The flask contents are again taken to dryness under reduced pressure at 40 ° . This last procedure is repeated twice more to strip the [85S]MPR and [35S]rhombic sulfur of residual pyridine. The stripping procedure is important because [s~S]rhombic sulfur dissolves in pyridine, and any pyridine remaining will carry along the sulfur in subsequent purification steps. The [~S]MPR is dissolved in the flask with 15 ml of hot water and filtered while hot through a sintered-glass funnel with a fine filter and a suction flask. The [3~S]MPR which crystallizes from solution on cooling is redissolved with gentle heating and again filtered through a clean, fine sintered-glass funnel. The [85S]MPR is again dissolved and dried in the same manner. The dried [~sS]MPR is taken up with i0 ml of hot water and filtered while hot through a clean, fine sintered-glass funnel. The solution in a 20-ml beaker is allowed to crystallize in the refrigerator overnight. Crystals of [s~S]MPR are collected and dried under reduced pressure to yield about 220 mg (77%) of ~99% radiopure compound. For preparation of the 6-methyl[35S]thio derivative (6-methyl[85S]thioinosine, [85S]MTI), 0.775 mmole (220 mg) of [85S]MPR is allowed to react with 0.787 mmole (0.049 ml, 1.015 equivalents) of methyliodide in 1.67 ml of 0.4 N NaOH with constant stirring for 10 min. Then 0.27 ml of 0.4 N NaOH and 0.049 ml of methyliodide are added and constantly stirred for an additional 10 rain. The reaction mixture is maintained at L. L. Bennett, Jr., R. W. Brockman, H. P. Schneibli, S. Chumley, G. J. Dixon, F. M. Schabel, Jr., E. A. Dulmadge, H. E. Skipper, J. A. Montgomery, and H. J. Thomas, Nature (London) 205, 1276 (1965).
[37]
DNA-DEPENDENT RNA POLYMERASE
355
room temperature for several hours, seeded with a few crystals of unlabeled MTI, and placed in the refrigerator overnight to crystallize. The crystals of [~S]MTI (190-200 mg) are collected, dried, and refluxed with 1.0 ml of absolute ethanol for several minutes and filtered through a fine sintered-glass funnel with suction while hot. It is important here to maintain anhydrous conditions since [3~S]MTI is very soluble in ethanol containing traces of water. The crystals are dried under reduced pressure. The yield of [3SS]MTI is 140-150 mg. Periodate oxidation to give the final product, [3~S]MMPR-0P, is carried out as follows. [3~S]MTI, 0.475 mmole (142 rag), is allowed to react with 0.52 mmole (120 rag, 1.1 equivalents) of periodic acid in 5.20 ml of water in the dark for 30 min. The reaction mixture is pipetted onto a Dowex 1-formate column, 10 X 30 mm, and the eluate and two washes are collected in a 50-ml round-bottom flask and immediately lyophilized to yield about 100 mg of pure [85S]MMPR-OP. The initial specific activity is about 7000 cpm/nmole. All these reactions may be scaled up or down, as long as the correct proportions described here are held constant. Affinity Labeling DNA-Dependent R N A Polymerase An affinity label has an affinity for the most reactive site on the enzyme; however, other sites will be bound at high label concentrations. Equation (1) can be used to determine the precise concentration [3~S]M M P R - O P that binds to the active center of RNA polymerase in a 1 : 1 molar ratio. Here, (AL. E ) / E = KAL
(I)
A L . E is the molar concentration of the [3sS] M M P R - O P - R N A polymerase complex, E is the total molar enzyme concentration, AL is the molar concentration of [35S]MMPR-OP, and K is the binding constant at equilibrium. Taking the log of both sides log (AL- E ) / E = log KAL
(2)
and rearranging to give log (AL • E ) / E = log K 4- log AL
(3)
a plot of log AL. E / E vs. log AL gives a straight line. The point at which the line passes through zero is the concentration of [35S]MMPR-OP that binds to RNA polymerase in a 1 : 1 molar ratio since log (AL. E ) / E -- log 0 and the antilog of zero is 1.
(4)
356
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[37]
DETERMINATION OF THE CONCENTRATION OF METHYLTHIOINOSINEDICARBOXALDEHYDE WHICH BINDS IN A 1 : 1 M O L A R RATIO WITH RNA POLYMERA.SE a
Concentration of affinity label 1 7.5 5 2.5. 1 5
× 10-3 X 10-' )< 10-4 )< 10-4 X 10-4 × 10-5
Log AL concentration
AL: Enzyme (ratio)
Log A L . E ratio
--3.00 --3.12 --3.30 --3.60 -4.00 -4.30
5.96 4.01 2.65 1.43 0.56 0.27
-}-0.78 +0.60 +0.42 +0.16 --0.25 --0.57
RNA polymerase (49 ~g per reaction mixture) was incubated for 15 min at 37 ° with varying concentratimls of [uS]MMPR-OP in Tris at pH 7.9, containing 50 ~,moles of Tris, 4 ~moles of MgC12, and 0.2 M with respect to KC1. No substrates or template were present. The reaction mixtures were cooled to 4 °, and the reactions were stopped by the addition of 5 % TCA. This treated mixture was filtered through 2.4-cm Whatman G F / C glass fiber filters, and the unbound radiolabelecl compound was washed through with 50 ml of 2% TCA and 3 ml of 95 % ethanol. The filters were allowed to air dry for 1 hr. Finally, they were placed directly into scintillation vials containing 10 ml of a toluene fluor and counted in a liquid scintillation spectrometer.
An application of this method and of Eq. (3) is shown in the table using 0.1 nmole of enzyme and varying concentrations of [35S]MMPR-0P. A plot of these data gave a concentration of 0.18 mM [ ~ S ] M M P R - 0 P , which would bind 0.1 nmole of enzyme in a 1:1 ratio. Higher concentrations of [85S]MMPR-OP begin to bind to lysines not in the active center. Pragmatically, concentrations of [asS]MMPR-0P less than 0.18 mM would have been used to label 0.1 nmole of enzyme if the stable amine bond were to be formed between [35S]MMPR-OP and the enzyme by sodium borohydride reduction. For instance, 1.36 nmoles of enzyme and 0.5 mM [85S]MMPR-OP were used to affinity label the lysine in the initiation subsite in the active center of RNA polymerase 2 (see below).
Enzyme Purification
Homogeneous preparations of Escher~chia coli DNA-dependent RNA polymerase are required for affinity labeling. The method of Burgess and Jendrisak can be used to prepare the core enzyme2 R. R. Burgess and J. J. Jendrisak, Biochemistry 14, 4634 (1975).
[37]
DNA-DEPENDENT RNA POLYMERA.SE
357
Binding o] [~S]MMPR-OP to RNA Polymerase: Formation o] the Schif] Base Binding to DNA-dependent RNA polymerase has been carried out as follows. Enzyme, 1.36 nmoles (679 ~g), was incubated for 45 rain at 37 ° with 0.5 mM [~SS]MMPR-OP in 1.0 ml of 1.0 M KHC03 at pH 7.9, which was 8 mM in MgCl~ and 0.2 M in KC1. Tris buffer does not work well here because [3~S]MMPR-OP reacts with the amino group of Tris upon sodium borohydride reduction (see below). This prohibition probably holds for any buffer containing reactive amino groups if sodium borohydride reduction is to be used.
Reduction o] the Schi]] Base to the Stable Amine The Schiff base is reduced to the amine bond by mild reduction with sodium borohydride (NaBH4). Immediately after formation of the Schiff base in the above reaction, the reaction mixture is cooled to 4 ° in ice and 15 mg of NaBH4 in 1.0 ml of KHC03 at pH 7.9 is added. The reduction is allowed to occur in a cold-room at 4 ° for 12 hr. Since the reaction liberates hydrogen gas, reaction tubes are loosely capped to avoid a buildup of pressure. Unreacted [35S]MMPR-OP and NaBH4 are removed by dialyzing the reaction mixture against excess KHC0~ at pH. 7.9 until the radioactivity in the buffer drops to background levels. Disk electrophoresis on polyacrylamide gels of the denatured enzyme[~SS]MMPR-OP complex is carried out to determine the subunit bound by the affinity label.:
Comments The [35S]MMPR-0P has recently been used to affinity label the active center of E. coli DNA-dependent DNA polymerase I. o The compound has also been used to label the lysine near the active center of ribonuclease A. 7 In all studies carried out to date on enzymes of nucleic acid metabolism, M M P R - O P has shown specificity for lysine residues. Inhibition of other enzymes of nucleic acid metabolism by M M P R - O P can be taken as presumptive evidence for the presence of lysines in their active R. A. Salvo, G. F. Serio, J. E. Evans, and A. P. Kimball, Biochemistry 15, 493 (1976). ' T. C. Spoor, J. L. Hodnett, and A. P. Kimball, Cancer Res. 33, 856 (1973).
358
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[38]
centers, and the procedures described above could be modified for their study. Dowex 1-formate columns should be washed with concentrated formic acid and then eluted with distilled water until neutral before use for purifying MMPR-OP. The formic acid wash produces a "milky" solution at the solvent front that would otherwise contaminate the MMPR-0P. The 6-methyl-[14C]MMPR-OP could be prepared by substituting [~4C]methyliodide in the methylation step. This compound has the advantage of a long half-life; [85S]MMPR-OP has a half-life of about 3 months. [a~S]Rhombic sulfur is usually supplied in a solution of benzene. The solution is placed in the round-bottom flask in which the sulfur exchange is to be carried out, then the benezene is removed by distillation. Care should be exercised to avoid polymerizing or subliming the [35S]rhombic sulfur.
[38] S t a p h y l o c o c c a l N u c l c a s e By PEDRO CUATRECASASand MEIR WILCHEK Mierococcal nuclease is an extracellular enzyme of Staphylococcus aureus that hydrolyzes specific phosphodiester bonds of both RNA and DNA. 1 The protein is a convenient model for studies of conformational stability, effects of ligand binding, chemical modification, hydrolytic mechanism, and X-ray crystallographic analyses. 1 Two groups of reagents were used for affinity labeling of this enzyme, bromoacetyl (Fig. 1, reagents I-III), 2 and diazonium salts (Fig. 1, reagents IV-VI 3,4) of different thymidine-p-aminophenylphosphate derivatives. Reagents I and IV are derived from pdTp-aminophenyl, 5 which is a potent competitive inhibitor of the enzyme, having a dissociation con1 p. Cuatrecasas, H. Taniuchi, and C. B. Anfinsen, Brookhaven Symp. Biol. 21,
172 (1969). 2 p. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, J. Biol. Chem. 244, 4316 (1969). 3 p. Cuatrecasas, J. Biol. Chem. 295, 574 (1970). ' M. Wilchek, FEBS Lett. 7, 161 (1970). 5 Abbreviations: pdTp, deoxythymidine 3',5'-diphosphate; pdTp-nitrophenyl, deoxythymidine 3'-p-nitrophenylphosphate 5'-phosphate; nitrophenyl-pdT, p-nitrophenyl ester of deoxythymidine 5'-phosphate; dTp-nitrophenyl, p-nitrophenyl ester of deoxythymidine 3'-phosphate ; dTp-aminophenyl, deoxythymidine 3'-p-aminophenylphosphate; pdTp-aminophenyl, deoxythymidine 3'-p-aminophenylphosphate 5'-phosphate; aminophenyl-pdT, deoxythymidine 5'-p-aminophenylphosphate.
DNA-DEPENDENT RNA POLYMERASE
[37] D N A - D e p e n d e n t
353
RNA Polymerase 1
By A. P. KIMBALL An essential lysine residue in the active center of the catalytic subunit of DNA-dependent RNA polymerase can be affinity labeled with methylthioinosinedicarboxaldehyde (MMPR-0p).2, 3 The c-amino group of the lysine forms a Schiff base with an aldehyde group of MMPR-OP, which can be converted to the stable amine by mild reduction with sodium borohydride. The [8~S]MMPR-OP can be prepared easily for radioactive affinity labeling of the enzyme.
SCH3 HOCH2
[
MMPR-OP Preparation of Methylthioinosinedicarboxaldehyde (MMPR-OP) Synthesis o] Unlabeled MMPR-OP One millimole of 9-fl-~-ribofuranosyl-6-methylthiopurine (298 mg) (methylthioinosine, MTI) is allowed to react with 1.1 mmoles of periodic acid (H~106, 251 rag) in 11 ml of distilled water in the dark for 30 min. The solution is rapidly pipetted onto a Dowex 1-formate column (10 X 30 mm), and the self-eluate and two water washes are collected in a 100-ml round-bottom flask. The solution of MMPR-0P, approximately 30 mi, is frozen in a thin film around the sides of the flask by rotation in a Dry Ice-acetone mixture, and the frozen solution is immediately lyophilized to give a white fluffy powder. A yield of 70-80% can be expected. The MMPR-OP should be stored dry at --20 °. Solutions of 'This research was supported by research grants from the Robert A. Welch Foundation (E-321) and from the NCI (CA-12327) of National Institutes of Health. 2j. Nixon, T. Spoor, J. Evans, and A. P. Kimball, Biochemistry 11, 4570 (1972). 3T. C. Spoor, F, L. Persico, J. E. Evans, and A. P. Kimball, Nature (London) 227, 57 (1970).
354
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[37]
MMPR-OP should be prepared immediately before use. Any excess solution should be discarded since the MMPR-OP slowly degrades in solution to 6-methylthiopurine with a half-life of about 4 days. The MMPR-OP is moderately difficult to dissolve in water, but heat may not be used to aid solution because this rapidly degrades the MMPR-OP.
Synthesis o] [8~S]MMPR-OP One millimole (284 mg) of 9-fl-D-ribofuranosyl-6-thiopurine (thioinosine, mercaptopurine riboside, MPR) is allowed to react with 1 mmole (32 mg) of [35S]rhombic sulfur, 20 mCi/mmole, in 25 ml of dr)" pyridine under reflux for 4 hr. 4 Care should be taken to keep the MPR from accumulating around the wall of the round-bottom flask at the solution surface if a heating mantle is used, since charring may occur. After 4 hr of reflux, 25 ml of distilled water is added to the pyridine solution, and the solution is taken to dryness under reduced pressure at 40 ° . Water, 25 ml, is added to the flask and gently heated in a water bath to dissolve the [35S]MPR. A yellow-gray film that does not dissolve is [aSS]rhombic sulfur. The flask contents are again taken to dryness under reduced pressure at 40 ° . This last procedure is repeated twice more to strip the [85S]MPR and [35S]rhombic sulfur of residual pyridine. The stripping procedure is important because [s~S]rhombic sulfur dissolves in pyridine, and any pyridine remaining will carry along the sulfur in subsequent purification steps. The [~S]MPR is dissolved in the flask with 15 ml of hot water and filtered while hot through a sintered-glass funnel with a fine filter and a suction flask. The [3~S]MPR which crystallizes from solution on cooling is redissolved with gentle heating and again filtered through a clean, fine sintered-glass funnel. The [85S]MPR is again dissolved and dried in the same manner. The dried [~sS]MPR is taken up with i0 ml of hot water and filtered while hot through a clean, fine sintered-glass funnel. The solution in a 20-ml beaker is allowed to crystallize in the refrigerator overnight. Crystals of [s~S]MPR are collected and dried under reduced pressure to yield about 220 mg (77%) of ~99% radiopure compound. For preparation of the 6-methyl[35S]thio derivative (6-methyl[85S]thioinosine, [85S]MTI), 0.775 mmole (220 mg) of [85S]MPR is allowed to react with 0.787 mmole (0.049 ml, 1.015 equivalents) of methyliodide in 1.67 ml of 0.4 N NaOH with constant stirring for 10 min. Then 0.27 ml of 0.4 N NaOH and 0.049 ml of methyliodide are added and constantly stirred for an additional 10 rain. The reaction mixture is maintained at L. L. Bennett, Jr., R. W. Brockman, H. P. Schneibli, S. Chumley, G. J. Dixon, F. M. Schabel, Jr., E. A. Dulmadge, H. E. Skipper, J. A. Montgomery, and H. J. Thomas, Nature (London) 205, 1276 (1965).
[37]
DNA-DEPENDENT RNA POLYMERASE
355
room temperature for several hours, seeded with a few crystals of unlabeled MTI, and placed in the refrigerator overnight to crystallize. The crystals of [~S]MTI (190-200 mg) are collected, dried, and refluxed with 1.0 ml of absolute ethanol for several minutes and filtered through a fine sintered-glass funnel with suction while hot. It is important here to maintain anhydrous conditions since [3~S]MTI is very soluble in ethanol containing traces of water. The crystals are dried under reduced pressure. The yield of [3SS]MTI is 140-150 mg. Periodate oxidation to give the final product, [3~S]MMPR-0P, is carried out as follows. [3~S]MTI, 0.475 mmole (142 rag), is allowed to react with 0.52 mmole (120 rag, 1.1 equivalents) of periodic acid in 5.20 ml of water in the dark for 30 min. The reaction mixture is pipetted onto a Dowex 1-formate column, 10 X 30 mm, and the eluate and two washes are collected in a 50-ml round-bottom flask and immediately lyophilized to yield about 100 mg of pure [85S]MMPR-OP. The initial specific activity is about 7000 cpm/nmole. All these reactions may be scaled up or down, as long as the correct proportions described here are held constant. Affinity Labeling DNA-Dependent R N A Polymerase An affinity label has an affinity for the most reactive site on the enzyme; however, other sites will be bound at high label concentrations. Equation (1) can be used to determine the precise concentration [3~S]M M P R - O P that binds to the active center of RNA polymerase in a 1 : 1 molar ratio. Here, (AL. E ) / E = KAL
(I)
A L . E is the molar concentration of the [3sS] M M P R - O P - R N A polymerase complex, E is the total molar enzyme concentration, AL is the molar concentration of [35S]MMPR-OP, and K is the binding constant at equilibrium. Taking the log of both sides log (AL- E ) / E = log KAL
(2)
and rearranging to give log (AL • E ) / E = log K 4- log AL
(3)
a plot of log AL. E / E vs. log AL gives a straight line. The point at which the line passes through zero is the concentration of [35S]MMPR-OP that binds to RNA polymerase in a 1 : 1 molar ratio since log (AL. E ) / E -- log 0 and the antilog of zero is 1.
(4)
356
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[37]
DETERMINATION OF THE CONCENTRATION OF METHYLTHIOINOSINEDICARBOXALDEHYDE WHICH BINDS IN A 1 : 1 M O L A R RATIO WITH RNA POLYMERA.SE a
Concentration of affinity label 1 7.5 5 2.5. 1 5
× 10-3 X 10-' )< 10-4 )< 10-4 X 10-4 × 10-5
Log AL concentration
AL: Enzyme (ratio)
Log A L . E ratio
--3.00 --3.12 --3.30 --3.60 -4.00 -4.30
5.96 4.01 2.65 1.43 0.56 0.27
-}-0.78 +0.60 +0.42 +0.16 --0.25 --0.57
RNA polymerase (49 ~g per reaction mixture) was incubated for 15 min at 37 ° with varying concentratimls of [uS]MMPR-OP in Tris at pH 7.9, containing 50 ~,moles of Tris, 4 ~moles of MgC12, and 0.2 M with respect to KC1. No substrates or template were present. The reaction mixtures were cooled to 4 °, and the reactions were stopped by the addition of 5 % TCA. This treated mixture was filtered through 2.4-cm Whatman G F / C glass fiber filters, and the unbound radiolabelecl compound was washed through with 50 ml of 2% TCA and 3 ml of 95 % ethanol. The filters were allowed to air dry for 1 hr. Finally, they were placed directly into scintillation vials containing 10 ml of a toluene fluor and counted in a liquid scintillation spectrometer.
An application of this method and of Eq. (3) is shown in the table using 0.1 nmole of enzyme and varying concentrations of [35S]MMPR-0P. A plot of these data gave a concentration of 0.18 mM [ ~ S ] M M P R - 0 P , which would bind 0.1 nmole of enzyme in a 1:1 ratio. Higher concentrations of [85S]MMPR-OP begin to bind to lysines not in the active center. Pragmatically, concentrations of [asS]MMPR-0P less than 0.18 mM would have been used to label 0.1 nmole of enzyme if the stable amine bond were to be formed between [35S]MMPR-OP and the enzyme by sodium borohydride reduction. For instance, 1.36 nmoles of enzyme and 0.5 mM [85S]MMPR-OP were used to affinity label the lysine in the initiation subsite in the active center of RNA polymerase 2 (see below).
Enzyme Purification
Homogeneous preparations of Escher~chia coli DNA-dependent RNA polymerase are required for affinity labeling. The method of Burgess and Jendrisak can be used to prepare the core enzyme2 R. R. Burgess and J. J. Jendrisak, Biochemistry 14, 4634 (1975).
[37]
DNA-DEPENDENT RNA POLYMERA.SE
357
Binding o] [~S]MMPR-OP to RNA Polymerase: Formation o] the Schif] Base Binding to DNA-dependent RNA polymerase has been carried out as follows. Enzyme, 1.36 nmoles (679 ~g), was incubated for 45 rain at 37 ° with 0.5 mM [~SS]MMPR-OP in 1.0 ml of 1.0 M KHC03 at pH 7.9, which was 8 mM in MgCl~ and 0.2 M in KC1. Tris buffer does not work well here because [3~S]MMPR-OP reacts with the amino group of Tris upon sodium borohydride reduction (see below). This prohibition probably holds for any buffer containing reactive amino groups if sodium borohydride reduction is to be used.
Reduction o] the Schi]] Base to the Stable Amine The Schiff base is reduced to the amine bond by mild reduction with sodium borohydride (NaBH4). Immediately after formation of the Schiff base in the above reaction, the reaction mixture is cooled to 4 ° in ice and 15 mg of NaBH4 in 1.0 ml of KHC03 at pH 7.9 is added. The reduction is allowed to occur in a cold-room at 4 ° for 12 hr. Since the reaction liberates hydrogen gas, reaction tubes are loosely capped to avoid a buildup of pressure. Unreacted [35S]MMPR-OP and NaBH4 are removed by dialyzing the reaction mixture against excess KHC0~ at pH. 7.9 until the radioactivity in the buffer drops to background levels. Disk electrophoresis on polyacrylamide gels of the denatured enzyme[~SS]MMPR-OP complex is carried out to determine the subunit bound by the affinity label.:
Comments The [35S]MMPR-0P has recently been used to affinity label the active center of E. coli DNA-dependent DNA polymerase I. o The compound has also been used to label the lysine near the active center of ribonuclease A. 7 In all studies carried out to date on enzymes of nucleic acid metabolism, M M P R - O P has shown specificity for lysine residues. Inhibition of other enzymes of nucleic acid metabolism by M M P R - O P can be taken as presumptive evidence for the presence of lysines in their active R. A. Salvo, G. F. Serio, J. E. Evans, and A. P. Kimball, Biochemistry 15, 493 (1976). ' T. C. Spoor, J. L. Hodnett, and A. P. Kimball, Cancer Res. 33, 856 (1973).
358
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[38]
centers, and the procedures described above could be modified for their study. Dowex 1-formate columns should be washed with concentrated formic acid and then eluted with distilled water until neutral before use for purifying MMPR-OP. The formic acid wash produces a "milky" solution at the solvent front that would otherwise contaminate the MMPR-0P. The 6-methyl-[14C]MMPR-OP could be prepared by substituting [~4C]methyliodide in the methylation step. This compound has the advantage of a long half-life; [85S]MMPR-OP has a half-life of about 3 months. [a~S]Rhombic sulfur is usually supplied in a solution of benzene. The solution is placed in the round-bottom flask in which the sulfur exchange is to be carried out, then the benezene is removed by distillation. Care should be exercised to avoid polymerizing or subliming the [35S]rhombic sulfur.
[38] S t a p h y l o c o c c a l N u c l c a s e By PEDRO CUATRECASASand MEIR WILCHEK Mierococcal nuclease is an extracellular enzyme of Staphylococcus aureus that hydrolyzes specific phosphodiester bonds of both RNA and DNA. 1 The protein is a convenient model for studies of conformational stability, effects of ligand binding, chemical modification, hydrolytic mechanism, and X-ray crystallographic analyses. 1 Two groups of reagents were used for affinity labeling of this enzyme, bromoacetyl (Fig. 1, reagents I-III), 2 and diazonium salts (Fig. 1, reagents IV-VI 3,4) of different thymidine-p-aminophenylphosphate derivatives. Reagents I and IV are derived from pdTp-aminophenyl, 5 which is a potent competitive inhibitor of the enzyme, having a dissociation con1 p. Cuatrecasas, H. Taniuchi, and C. B. Anfinsen, Brookhaven Symp. Biol. 21,
172 (1969). 2 p. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, J. Biol. Chem. 244, 4316 (1969). 3 p. Cuatrecasas, J. Biol. Chem. 295, 574 (1970). ' M. Wilchek, FEBS Lett. 7, 161 (1970). 5 Abbreviations: pdTp, deoxythymidine 3',5'-diphosphate; pdTp-nitrophenyl, deoxythymidine 3'-p-nitrophenylphosphate 5'-phosphate; nitrophenyl-pdT, p-nitrophenyl ester of deoxythymidine 5'-phosphate; dTp-nitrophenyl, p-nitrophenyl ester of deoxythymidine 3'-phosphate ; dTp-aminophenyl, deoxythymidine 3'-p-aminophenylphosphate; pdTp-aminophenyl, deoxythymidine 3'-p-aminophenylphosphate 5'-phosphate; aminophenyl-pdT, deoxythymidine 5'-p-aminophenylphosphate.
