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[28] A method for anaerobic column chromatography
322
[28] A M e t h o d
CHROMATOGRAPHIC PROCEDURES
for Anaerobic
Column
[28]
Chromatography
By R. REPASKE Column chromatography of an oxygen-sensitive enzyme (hydrogenase) was accomplished with more than 90% recovery using the anaerobic technique described. Previously, very low recovery was obtained by the usual method of incorporating reducing agent in the elution buffer. The anaerobic system consisted of a column and an elution buffer vessel, both modified to facilitate anaerobic assembly and operation. During operation the volume of eluted fractions removed from the system was balanced by an equivalent input of an oxygen-free gas. The system was assembled from individual units (Fig. 1; units A, B, and C) from which air was flushed with O2-free gas. These units could be opened subsequently for making additions or for making connections between units if a sufficiently large outflow of gas was maintained to create an anaerobic atmosphere at the opening. The anaerobic system could be assembled, operated, and maintained with low levels of contaminating oxygen if attention was given to small details such as keeping a slight positive pressure in the system and flushing capillary tubing with gas before making the terminal connection. Multiple gas lines were required for flushing several parts of the system simultaneously. For most purposes large volumes of gas were needed and the usual cylinder gas regulator gave adequate control. However, while collecting fractions, small volumes of gas at very low pressure were required. It was essential to insert a reducing valve capable of delivering small volumes of gas at a few ounces positive pressure. Connections between units were made with polyethylene capillary tubing having female Kel-F Luer fittings which connected with male Luer ends on the glass tubing.
Anaerobic Buffers and Sorbent Anaerobic buffer solutions needed for elution and for packing the column were prepared first. Eluent buffers were placed in unit B (Fig. 1), and additional buffer to be used to pack the column bed was placed in a reagent bottle. (Reagent bottles with bottom tubulation could be used in place of unit B.) The tops for unit B had a long-stemmed glass tube for bubbling Oz-free gas through the liquid and a short-stemmed vent tube. The same tube arrangement in a rubber stopper was used with reagent bottles. For convenience the tubes were provided with stopcocks for quick closure. Reducing agent was introduced shortly before
[28]
ANAEROBIC COLUMN CHROMATOGRAPHY
C
323
i ,il
~"",:/,D8 m FIo. 1. Schematic drawing of the assembled anaerobic column system. A, a plastic bottle which receives low-pressure O2-free gas through stopcock a; B, a Plexiglas twochamber eluent buffer vessel for either step or for gradient elutions; C, the column; and D, a serum bottle containing a collected fraction being vented with O2-free gas.
gassing was completed (20 minutes) by injecting the solution through the serum bottle stopper (J) in the lid of B or by raising the reagent bottle stoppers; escaping gas formed an adequate seal against air. Buffer equilibrated sorbent slurry was also placed in a reagent bottle and was made anaerobic as described above. The Plexiglas eluent buffer vessel (B) consisted of two identical compartments which were used separately for step elutions or together for gradient elutions. With gradients, the connection above the liquid was needed to maintain equal pressure in both compartments. The long tube used to add O2-free gas to the buffer also functioned as a Mariotte tube during operation of the column. The screw cap covers had an 'O' ring seal to provide an air-tight closure. Anaerobic Column
The column (C) was a modified commercially available column. Immediately below the rubber stopper a small-diameter glass tubing enters
324
CHROMATOGRAPHIC PROCEDURES
[28]
the column, its open end directed downward. O2-free gas admitted to the column through this tube was used for flushing the column and for providing a protective baffle of gas at the top through which anaerobic buffer, sorbent slurry or enzyme could be added without exposure to air. Once the column had been made anaerobic and stoppered, it could be safely reopened if gas was simultaneously admitted through stopcock j. The bottom member of the column, a male standard taper joint with a sintered-glass disk support, was modified by replacing the drip tube with a 12-ml conical centrifuge tube. Stopcocks were located as shown (k, l, and m). The column was made anaerobic by flushing gas for 15-20 minutes through b o t h j (with stopper removed) and k; gas exhaust in the bottom section was alternated between l and m. Stopcock k and the exhaust stopcock were closed whilej remained open and anaerobic buffer and sorbent slurry were poured through the gas flowing out of the top of the column. Because the bottom portion of the column was closed, buffer did not pass through to the collecting tube. Buffer flow required for packing the sorbent to form the column bed was controlled by releasing pressure through 1. As a precaution against air entering the open stopcock, gas flow was maintained by simultaneously opening stopcock k. With subsequent additions of buffer or sorbent, liquid flow was controlled by opening or closing these stopcocks. (It should be noted that each time bottles containingbuffer or sorbent were opened, it was necessary to flush them again with O2-free gas.) The top of the column was sealed with a solid rubber stopper as stopcock j was closed.
Assembly of Components Unit B was next connected to the low-pressure Oz-free gas source. The gas was connected through a to the plastic bottle A, which acted as a safety buffer against sudden pressure changes. A, each of the stopcocks (b, c), and the capillary tubing were flushed before the tubing was connected to d. Small amounts of air in the glass tubing before stopcock d were vented through B via h (or e if one elution buffer was used) and through the capillary tubing to be connected to the bottom part of the column. After about 5 minutes the tubing was connected to stopcock k and venting was continued for a time through l. Stopcocks k and l and the gas source were then closed. Air in tubing n was displaced with anaerobic buffer from B.
Operation of the Column Enzyme was now added to the column after removal of the stopper with a protective flow of gas from j; enzyme was washed in with anaer-
[29]
C H R O M A T O G R A P H Y OF PROTEINS
325
obic buffer by controlling c o l u m n flow with k a n d / as described above. T h e s t o p p e r with tubing n was placed on the c o l u m n a s j was closed. T h e buffer flow rate was established a n d the gas p r e s s u r e was adjusted so that each d r o p o f buffer caused a gas bubble to rise f r o m the Mariotte tube. W h e n the system was completely assembled, stopcock k h a d to be o p e n e d to p r o v i d e p r e s s u r e equalization between B a n d the collecting tube if the c o l u m n was to flow. W h e n a g r a d i e n t elution was used, stopcocks g a n d i were o p e n e d and the magnetic stirrer was started. Fractions o f the desired v o l u m e were collected a n d d i s p e n s e d (m) into preflushed s e r u m bottles while Oz-free gas was directed into the bottle. A s e r u m bottle s t o p p e r sealed the bottle a n d gas was flushed t h r o u g h the head space a n d vented by h y p o d e r m i c needles (D).
[29] Chromatography of Proteins on Hydroxyapatite By GIORGIO BERNARDI
C h r o m a t o g r a p h y o f proteins on h y d r o x y a p a t i t e t ( H A 2) columns is a separation technique d e v e l o p e d in Tiselius' l a b o r a t o r y ? -~ T h e use o f H A as p r e p a r e d by Tiselius et al. 4-6 has s u p e r s e d e d that o f o t h e r calcium p h o s p h a t e s previously e m p l o y e d in protein p u r i f i c a t i o n / T h e relatively slow acceptance o f c h r o m a t o g r a p h y on H A c o l u m n s by p r o t e i n chemists seems to be d u e largely to t h r e e m a i n factors: the r a t h e r laborious p r e p a r a t i o n p r o c e d u r e ; the u n k n o w n m e c h a n i s m o f interaction o f proteins with HA; the introduction o f cellulose ion-exchangers 1Hydroxyapatite, not hydroxylapatite, is the name recommended by Wyckoff34 since "hydroxyl" implies the derivatives being named after the subtituted ion, a usage which is not observed in the corresponding fluorine and chlorine derivatives (e.g., fluorapatite, chlorapatite, not fluoridapatite, chloridapatite). 2Abbreviations: HA, hydroxyapatite; NAP, KP, equimolar mixtures of NaH2PO4, Na2_ HPO4, and KH~PO4, K2HPO4, respectively. The pH is close to 6.8, and the ionic strength is equal to about twice the molarity. The abbreviation PB (phosphate buffer) used by some authors does not indicate the cation. Because the eluting power of phosphates is different for different salts, the abbreviation PB is discouraged. 3S. M. Swingle and A. Tiselius, Biochem.J. 48, 171 ( 1951). 4A. Tiselius, Ark. Kemi 7, 445 (1954). ~A. Tiselius, S. Hjert6n, and O. Levin,Arch. Biochem. Biophys. 65, 132 (1956). eS. Hjert~n, Biochim. Biophys. Acta 3 l, 216 ( !956). 70. Levin, this series, 5, 27. sC. A. Zittle, Advan. Enzymol. 14, 319 (1953).
[28] A M e t h o d
CHROMATOGRAPHIC PROCEDURES
for Anaerobic
Column
[28]
Chromatography
By R. REPASKE Column chromatography of an oxygen-sensitive enzyme (hydrogenase) was accomplished with more than 90% recovery using the anaerobic technique described. Previously, very low recovery was obtained by the usual method of incorporating reducing agent in the elution buffer. The anaerobic system consisted of a column and an elution buffer vessel, both modified to facilitate anaerobic assembly and operation. During operation the volume of eluted fractions removed from the system was balanced by an equivalent input of an oxygen-free gas. The system was assembled from individual units (Fig. 1; units A, B, and C) from which air was flushed with O2-free gas. These units could be opened subsequently for making additions or for making connections between units if a sufficiently large outflow of gas was maintained to create an anaerobic atmosphere at the opening. The anaerobic system could be assembled, operated, and maintained with low levels of contaminating oxygen if attention was given to small details such as keeping a slight positive pressure in the system and flushing capillary tubing with gas before making the terminal connection. Multiple gas lines were required for flushing several parts of the system simultaneously. For most purposes large volumes of gas were needed and the usual cylinder gas regulator gave adequate control. However, while collecting fractions, small volumes of gas at very low pressure were required. It was essential to insert a reducing valve capable of delivering small volumes of gas at a few ounces positive pressure. Connections between units were made with polyethylene capillary tubing having female Kel-F Luer fittings which connected with male Luer ends on the glass tubing.
Anaerobic Buffers and Sorbent Anaerobic buffer solutions needed for elution and for packing the column were prepared first. Eluent buffers were placed in unit B (Fig. 1), and additional buffer to be used to pack the column bed was placed in a reagent bottle. (Reagent bottles with bottom tubulation could be used in place of unit B.) The tops for unit B had a long-stemmed glass tube for bubbling Oz-free gas through the liquid and a short-stemmed vent tube. The same tube arrangement in a rubber stopper was used with reagent bottles. For convenience the tubes were provided with stopcocks for quick closure. Reducing agent was introduced shortly before
[28]
ANAEROBIC COLUMN CHROMATOGRAPHY
C
323
i ,il
~"",:/,D8 m FIo. 1. Schematic drawing of the assembled anaerobic column system. A, a plastic bottle which receives low-pressure O2-free gas through stopcock a; B, a Plexiglas twochamber eluent buffer vessel for either step or for gradient elutions; C, the column; and D, a serum bottle containing a collected fraction being vented with O2-free gas.
gassing was completed (20 minutes) by injecting the solution through the serum bottle stopper (J) in the lid of B or by raising the reagent bottle stoppers; escaping gas formed an adequate seal against air. Buffer equilibrated sorbent slurry was also placed in a reagent bottle and was made anaerobic as described above. The Plexiglas eluent buffer vessel (B) consisted of two identical compartments which were used separately for step elutions or together for gradient elutions. With gradients, the connection above the liquid was needed to maintain equal pressure in both compartments. The long tube used to add O2-free gas to the buffer also functioned as a Mariotte tube during operation of the column. The screw cap covers had an 'O' ring seal to provide an air-tight closure. Anaerobic Column
The column (C) was a modified commercially available column. Immediately below the rubber stopper a small-diameter glass tubing enters
324
CHROMATOGRAPHIC PROCEDURES
[28]
the column, its open end directed downward. O2-free gas admitted to the column through this tube was used for flushing the column and for providing a protective baffle of gas at the top through which anaerobic buffer, sorbent slurry or enzyme could be added without exposure to air. Once the column had been made anaerobic and stoppered, it could be safely reopened if gas was simultaneously admitted through stopcock j. The bottom member of the column, a male standard taper joint with a sintered-glass disk support, was modified by replacing the drip tube with a 12-ml conical centrifuge tube. Stopcocks were located as shown (k, l, and m). The column was made anaerobic by flushing gas for 15-20 minutes through b o t h j (with stopper removed) and k; gas exhaust in the bottom section was alternated between l and m. Stopcock k and the exhaust stopcock were closed whilej remained open and anaerobic buffer and sorbent slurry were poured through the gas flowing out of the top of the column. Because the bottom portion of the column was closed, buffer did not pass through to the collecting tube. Buffer flow required for packing the sorbent to form the column bed was controlled by releasing pressure through 1. As a precaution against air entering the open stopcock, gas flow was maintained by simultaneously opening stopcock k. With subsequent additions of buffer or sorbent, liquid flow was controlled by opening or closing these stopcocks. (It should be noted that each time bottles containingbuffer or sorbent were opened, it was necessary to flush them again with O2-free gas.) The top of the column was sealed with a solid rubber stopper as stopcock j was closed.
Assembly of Components Unit B was next connected to the low-pressure Oz-free gas source. The gas was connected through a to the plastic bottle A, which acted as a safety buffer against sudden pressure changes. A, each of the stopcocks (b, c), and the capillary tubing were flushed before the tubing was connected to d. Small amounts of air in the glass tubing before stopcock d were vented through B via h (or e if one elution buffer was used) and through the capillary tubing to be connected to the bottom part of the column. After about 5 minutes the tubing was connected to stopcock k and venting was continued for a time through l. Stopcocks k and l and the gas source were then closed. Air in tubing n was displaced with anaerobic buffer from B.
Operation of the Column Enzyme was now added to the column after removal of the stopper with a protective flow of gas from j; enzyme was washed in with anaer-
[29]
C H R O M A T O G R A P H Y OF PROTEINS
325
obic buffer by controlling c o l u m n flow with k a n d / as described above. T h e s t o p p e r with tubing n was placed on the c o l u m n a s j was closed. T h e buffer flow rate was established a n d the gas p r e s s u r e was adjusted so that each d r o p o f buffer caused a gas bubble to rise f r o m the Mariotte tube. W h e n the system was completely assembled, stopcock k h a d to be o p e n e d to p r o v i d e p r e s s u r e equalization between B a n d the collecting tube if the c o l u m n was to flow. W h e n a g r a d i e n t elution was used, stopcocks g a n d i were o p e n e d and the magnetic stirrer was started. Fractions o f the desired v o l u m e were collected a n d d i s p e n s e d (m) into preflushed s e r u m bottles while Oz-free gas was directed into the bottle. A s e r u m bottle s t o p p e r sealed the bottle a n d gas was flushed t h r o u g h the head space a n d vented by h y p o d e r m i c needles (D).
[29] Chromatography of Proteins on Hydroxyapatite By GIORGIO BERNARDI
C h r o m a t o g r a p h y o f proteins on h y d r o x y a p a t i t e t ( H A 2) columns is a separation technique d e v e l o p e d in Tiselius' l a b o r a t o r y ? -~ T h e use o f H A as p r e p a r e d by Tiselius et al. 4-6 has s u p e r s e d e d that o f o t h e r calcium p h o s p h a t e s previously e m p l o y e d in protein p u r i f i c a t i o n / T h e relatively slow acceptance o f c h r o m a t o g r a p h y on H A c o l u m n s by p r o t e i n chemists seems to be d u e largely to t h r e e m a i n factors: the r a t h e r laborious p r e p a r a t i o n p r o c e d u r e ; the u n k n o w n m e c h a n i s m o f interaction o f proteins with HA; the introduction o f cellulose ion-exchangers 1Hydroxyapatite, not hydroxylapatite, is the name recommended by Wyckoff34 since "hydroxyl" implies the derivatives being named after the subtituted ion, a usage which is not observed in the corresponding fluorine and chlorine derivatives (e.g., fluorapatite, chlorapatite, not fluoridapatite, chloridapatite). 2Abbreviations: HA, hydroxyapatite; NAP, KP, equimolar mixtures of NaH2PO4, Na2_ HPO4, and KH~PO4, K2HPO4, respectively. The pH is close to 6.8, and the ionic strength is equal to about twice the molarity. The abbreviation PB (phosphate buffer) used by some authors does not indicate the cation. Because the eluting power of phosphates is different for different salts, the abbreviation PB is discouraged. 3S. M. Swingle and A. Tiselius, Biochem.J. 48, 171 ( 1951). 4A. Tiselius, Ark. Kemi 7, 445 (1954). ~A. Tiselius, S. Hjert6n, and O. Levin,Arch. Biochem. Biophys. 65, 132 (1956). eS. Hjert~n, Biochim. Biophys. Acta 3 l, 216 ( !956). 70. Levin, this series, 5, 27. sC. A. Zittle, Advan. Enzymol. 14, 319 (1953).