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A modified method for determining hydrogen-deuterium exchange in proteins
SHORT COMMUNICATIONS
551
BBA 3 3 2 3 1
A modified method for determining hydrogen-deuterium exchange in proteins In the classical density-gradient method introduced by HVIDT et al. 1 for determining the hydrogen-deuterium exchange in proteins, the substances were subjected to three lyophilizations before the final measurements were performed. Although the reproducibility was high, the freeze-drying techniques involved could give distorted data and not all proteins could stand the severe treatment s. In this respect it was an essential improvement when the infrared spectrophotometric technique was introduced ~ which only required a single lyophilization, i.e. the initial one which removes the water before 2H20 is added. However, not all proteins can stand even this treatment, and for very labile proteins the hydrogen-tritium exchange method introduced by ENGLANDER4 is therefore preferred. In this method the exchangeable hydrogens are initially labeled with tritium b y incubating the protein in tritiated water. A Sephadex column is used to separate the tritiated protein from the tritiated solvent, and the back exchange of tritium from protein to ordinary solvent is measured in other Sephadex columns by separating protein from the tritiated solvent, and determining both protein concentration and residually bound tritium. In the present communication it is demonstrated that the gel filtration technique also can be used to measure the hydrogen-deuterium exchange in proteins, thus avoiding the lyophilization step normally required for the infrared spectrophotometric technique. The column used in the present study is approx. 0. 4 cm × 7 cm, Sephadex G-25, fine grade, which has been allowed to swell in ~H20. A matched pair of CaF 2 cells of o. I - m m path length was used, one cell always being used as the reference cell and the other as the sample cell. The outlet from the column was connected over a stopcock to the CaF~ sample cell by means of a thin teflon tubing. The top of the column could be connected to an external pressure source. A Perkin-Elmer spectrophotometer, Model I3U, was used to follow the disappearance of the amide I I band as the peptide hydrogen atoms exchanged with deuterium atoms. The instrument settings, operation procedures, and calculation of the data were similar to those previously described 5. A typical exchange experiment was carried out as follows: The reference CaF~ cell was filled with the appropriate 2H20 buffer solution. The Sephadex column was equilibrated with the same ~H~O buffer by passing 2 ml of the solution through the column connected to the sample cell. When the buffer in the column had drained down to the top of the Sephadex, IOO/~1 of an aqueous 6-8 % solution of the protein in H~O was transferred to the top of the column. The protein solution was quickly forced down into the Sephadex by means of the external air pressure, and this moment was taken as the start of the exchange. I ml of the zH~O buffer was quickly applied to the top of the column, and the elution was continued under air pressure. The amide I absorption was used to follow the elution of the protein. When the amide I absorption had reached the m a x i m u m value, the stopcock was closed in order to stop the flow through the cell. Frequent short scans were made alternating between the regions around the amide I I and the amide I bands, and the corresponding exchange times were noted. The first reading of amide I I could usually be made after 2-3 min. After Biochim. Biophys. Acta, 214 (197o) 551--553
552
SHORT COMMUNICATIONS
07
........... /]
0,6 I
tI
0.5,
o.: .~ o~ o,
Effluent ~/ol. (ml~
80
2
!20
20 Time (minl
60
Fig. I. Sephadex G-z5 column (in '2H~O) separation of ribonuclease from H~(). Column dimensions o. 4 c m x 8 cm. E l u e n t : o.o 5 M citrate buffer (P 2H 4.97) in 2H2(). Amide I a b s o r p t i o n was m e a s u r e d at 1648 c m - 1, and H20 a b s o r p t i o n was m e a s u r e d at the same wavelength. Fig. 2. Exchange-in curves lbr myoglobin (A) and ribonuclease (t3) at 2 1 . Myogiobin: exchange in o.o67 M p h o s p h a t e , Sephadex column technique (@), p~H 7.47, lyophilization technique (0) p=H 7.39; a m i d e I and a m i d e l l was m e a s u r e d at I 6 5 o c m a and I 5 4 7 c m ~, respectively. Ribonuclease: exchange in o . o 5 M p h o s p h a t e , Sephadex c o l u m n technique ( , , ) , p e r t 6 . 4 5 , lyophilization technique (0), pert 6.45; amide I and amide I I was measured at i~48 cm 1 and i543 cm -:x, respectively. The p=H values were calculated according to the equation of (;LAZOV: AND
LONG
6.
approx. 6 rain a complete scan of the spectrum starting from 175o cm ~ was performed, and additional scans were made after appropriate periods of exchange. The cells were removed from the beams in the spectrophotometer between the scans to minimize heating effects. When an exchange experiment was completed the column was cleaned by an additional 2 ml of 2H~O buffer. A total of 5-6 ml of~H20 was used per experiment. The results shown in Fig. I demonstrate that a complete separation of protein from water was obtained under these conditions. The HO2H absorption at I46O c m indicated that only traces of H20 were present in the protein peak in amounts insufficient to affect the amide I and amide II absorptions. Using the Sephadex column technique the hydrogen-deuterium exchange curves for ribonuclease and myoglobin were compared with results obtained using the traditional lyophilization technique ~. The results (Fig. 2) demonstrate that the two techniques gave identical curves confirming both the reliability of the Sephadex method, and lack of irreversible structural changes in ribonuclease and myoglobin after lyophilization. The gel filtration technique reported here can be used with advantage to study the hydrogen deuterium exchange of proteins which are unstable during lyophilization, provided the proteins are soluble in approx. 5- Io °',o concentration in an aqueous buffer, so that they have sufficient absorption in the amide I1 region after the dilution on the column. The author wants to thank Professor Martin Ottesen for his advice and support.
Carlsberg Laboratory, Chemical Department, Gl. Carlsbergvej zo, Valby, Copenhagen (Denmark) * Present address: Biophysics Research Boston, Mass. o2t 15 , U.S.A.
Biochim. Biophvs. Acta, 214 (107 o) 55t 55~,
Laboratory,
.I ACK T..} OHAYS~,:N"
Peter
Bent
Brigh~un Hospital,
SHORT COMMUNICATIONS
553
I A. HVIDT, (;. JOHANSEN AND K. LINDERSTROM-LANG (1960), in P. ALEXANDER AND R. J. BLOCK, A Laboratory Manual of Analytical Methods of Protein Chemistry, VoJ. 2, P e r g a m o n Press, Oxford, p. IOI. 2 A. HVlDT AND S. O. NIELSEN, Advan. Protein. Chem., 21 (1966) 287. 3 E. R. BLOUT, D. DE Loz~ AND A. ASADOURIAN,J. Am. Chem. Soc., 83 (1961) 1895. 4 S. W. ENGLANDER, Biochemistry, 2 (1963) 798. 5 L. WILLUMSEN, Compt. Rend. Tray. Lab. Carlsberg, 36 (1967) 247. 6 P. t{. GLAZOE AND F. A. LONG, J. Phys. Chem., 64 (196o) 188.
Received May I3th, 197o Biochim. Biophys. Acta, 214 (197 o) 551-553
BBA 3323O A note on the effect of dithionite on p-mercuribenzoate-reacted human hemoglobin Na~S204 is widely employed as a zeagent in hemoglobin research in spite of several reports suggesting that it m a y produce adverse side effects on the protein z-3. The main reason for the use of dithionite is its efficiency in completely and rapidly removing the oxygen in solution and in reducing ferric to ferrous heme. Dithionite side effects become more evident with dilute hemoglobin solutions and in the presence of a large excess of oxygen. When dithionite is added to hemoglobin solutions which have been prepared with deoxygenated buffer, in order to remove only the last traces of oxygen, control experiments show that the behavior of hemoglobin is not affected4, 5. In any case careful controls should always be carried out whenever dithionite is used. In the course of a study on human hemoglobin reacted stoichiometrically in /3-93 with p-mercuribenzoate (PMB), such controls revealed the occurrence of timedependent changes in the behavior of the PMB-treated hemoglobin due to the presence of dithionite, although air was rigorously excluded from the solutions. The changes appeared in both properties of PMB-treated hemoglobin we were studying: the kinetics of combination with CO after flash photolysis and the molecular weight of the CO derivative as measmed by differential gel filtration. In view of the wide use of both PMB and dithionite as hemoglobin reagents, it appeared worthwhile to repolt these experiments in the present note. It should be recalled that upon treatment of human hemoglobin with PMB only two SH groups (those at/3-93 ) react readily. The presence of PMB on/3-93 enhances the dissociation of the protein into dimers; on the other hand, the subsequent reaction of PMB with the other SH gloups (those at a-Io 4 and/3-112) promotes further dissociation into single a and/3 chainsT, 1°. H u m a n hemoglobin was prepared from freshly drawn blood b y the (NH4)2SO 4 method and was freed from organic and inorganic ions by passage through an ionexchange resin. Pzotein concentration was calculated on the basis of an extinction coefficient E ~ = 8.4 at 54 ° n m for the CO derivative. PMB, obtained commercially, Abbreviation : PMB, p-mercuribenzoate.
Biochim. Biophys. Acta, 214 (197 o) 553-555
551
BBA 3 3 2 3 1
A modified method for determining hydrogen-deuterium exchange in proteins In the classical density-gradient method introduced by HVIDT et al. 1 for determining the hydrogen-deuterium exchange in proteins, the substances were subjected to three lyophilizations before the final measurements were performed. Although the reproducibility was high, the freeze-drying techniques involved could give distorted data and not all proteins could stand the severe treatment s. In this respect it was an essential improvement when the infrared spectrophotometric technique was introduced ~ which only required a single lyophilization, i.e. the initial one which removes the water before 2H20 is added. However, not all proteins can stand even this treatment, and for very labile proteins the hydrogen-tritium exchange method introduced by ENGLANDER4 is therefore preferred. In this method the exchangeable hydrogens are initially labeled with tritium b y incubating the protein in tritiated water. A Sephadex column is used to separate the tritiated protein from the tritiated solvent, and the back exchange of tritium from protein to ordinary solvent is measured in other Sephadex columns by separating protein from the tritiated solvent, and determining both protein concentration and residually bound tritium. In the present communication it is demonstrated that the gel filtration technique also can be used to measure the hydrogen-deuterium exchange in proteins, thus avoiding the lyophilization step normally required for the infrared spectrophotometric technique. The column used in the present study is approx. 0. 4 cm × 7 cm, Sephadex G-25, fine grade, which has been allowed to swell in ~H20. A matched pair of CaF 2 cells of o. I - m m path length was used, one cell always being used as the reference cell and the other as the sample cell. The outlet from the column was connected over a stopcock to the CaF~ sample cell by means of a thin teflon tubing. The top of the column could be connected to an external pressure source. A Perkin-Elmer spectrophotometer, Model I3U, was used to follow the disappearance of the amide I I band as the peptide hydrogen atoms exchanged with deuterium atoms. The instrument settings, operation procedures, and calculation of the data were similar to those previously described 5. A typical exchange experiment was carried out as follows: The reference CaF~ cell was filled with the appropriate 2H20 buffer solution. The Sephadex column was equilibrated with the same ~H~O buffer by passing 2 ml of the solution through the column connected to the sample cell. When the buffer in the column had drained down to the top of the Sephadex, IOO/~1 of an aqueous 6-8 % solution of the protein in H~O was transferred to the top of the column. The protein solution was quickly forced down into the Sephadex by means of the external air pressure, and this moment was taken as the start of the exchange. I ml of the zH~O buffer was quickly applied to the top of the column, and the elution was continued under air pressure. The amide I absorption was used to follow the elution of the protein. When the amide I absorption had reached the m a x i m u m value, the stopcock was closed in order to stop the flow through the cell. Frequent short scans were made alternating between the regions around the amide I I and the amide I bands, and the corresponding exchange times were noted. The first reading of amide I I could usually be made after 2-3 min. After Biochim. Biophys. Acta, 214 (197o) 551--553
552
SHORT COMMUNICATIONS
07
........... /]
0,6 I
tI
0.5,
o.: .~ o~ o,
Effluent ~/ol. (ml~
80
2
!20
20 Time (minl
60
Fig. I. Sephadex G-z5 column (in '2H~O) separation of ribonuclease from H~(). Column dimensions o. 4 c m x 8 cm. E l u e n t : o.o 5 M citrate buffer (P 2H 4.97) in 2H2(). Amide I a b s o r p t i o n was m e a s u r e d at 1648 c m - 1, and H20 a b s o r p t i o n was m e a s u r e d at the same wavelength. Fig. 2. Exchange-in curves lbr myoglobin (A) and ribonuclease (t3) at 2 1 . Myogiobin: exchange in o.o67 M p h o s p h a t e , Sephadex column technique (@), p~H 7.47, lyophilization technique (0) p=H 7.39; a m i d e I and a m i d e l l was m e a s u r e d at I 6 5 o c m a and I 5 4 7 c m ~, respectively. Ribonuclease: exchange in o . o 5 M p h o s p h a t e , Sephadex c o l u m n technique ( , , ) , p e r t 6 . 4 5 , lyophilization technique (0), pert 6.45; amide I and amide I I was measured at i~48 cm 1 and i543 cm -:x, respectively. The p=H values were calculated according to the equation of (;LAZOV: AND
LONG
6.
approx. 6 rain a complete scan of the spectrum starting from 175o cm ~ was performed, and additional scans were made after appropriate periods of exchange. The cells were removed from the beams in the spectrophotometer between the scans to minimize heating effects. When an exchange experiment was completed the column was cleaned by an additional 2 ml of 2H~O buffer. A total of 5-6 ml of~H20 was used per experiment. The results shown in Fig. I demonstrate that a complete separation of protein from water was obtained under these conditions. The HO2H absorption at I46O c m indicated that only traces of H20 were present in the protein peak in amounts insufficient to affect the amide I and amide II absorptions. Using the Sephadex column technique the hydrogen-deuterium exchange curves for ribonuclease and myoglobin were compared with results obtained using the traditional lyophilization technique ~. The results (Fig. 2) demonstrate that the two techniques gave identical curves confirming both the reliability of the Sephadex method, and lack of irreversible structural changes in ribonuclease and myoglobin after lyophilization. The gel filtration technique reported here can be used with advantage to study the hydrogen deuterium exchange of proteins which are unstable during lyophilization, provided the proteins are soluble in approx. 5- Io °',o concentration in an aqueous buffer, so that they have sufficient absorption in the amide I1 region after the dilution on the column. The author wants to thank Professor Martin Ottesen for his advice and support.
Carlsberg Laboratory, Chemical Department, Gl. Carlsbergvej zo, Valby, Copenhagen (Denmark) * Present address: Biophysics Research Boston, Mass. o2t 15 , U.S.A.
Biochim. Biophvs. Acta, 214 (107 o) 55t 55~,
Laboratory,
.I ACK T..} OHAYS~,:N"
Peter
Bent
Brigh~un Hospital,
SHORT COMMUNICATIONS
553
I A. HVIDT, (;. JOHANSEN AND K. LINDERSTROM-LANG (1960), in P. ALEXANDER AND R. J. BLOCK, A Laboratory Manual of Analytical Methods of Protein Chemistry, VoJ. 2, P e r g a m o n Press, Oxford, p. IOI. 2 A. HVlDT AND S. O. NIELSEN, Advan. Protein. Chem., 21 (1966) 287. 3 E. R. BLOUT, D. DE Loz~ AND A. ASADOURIAN,J. Am. Chem. Soc., 83 (1961) 1895. 4 S. W. ENGLANDER, Biochemistry, 2 (1963) 798. 5 L. WILLUMSEN, Compt. Rend. Tray. Lab. Carlsberg, 36 (1967) 247. 6 P. t{. GLAZOE AND F. A. LONG, J. Phys. Chem., 64 (196o) 188.
Received May I3th, 197o Biochim. Biophys. Acta, 214 (197 o) 551-553
BBA 3323O A note on the effect of dithionite on p-mercuribenzoate-reacted human hemoglobin Na~S204 is widely employed as a zeagent in hemoglobin research in spite of several reports suggesting that it m a y produce adverse side effects on the protein z-3. The main reason for the use of dithionite is its efficiency in completely and rapidly removing the oxygen in solution and in reducing ferric to ferrous heme. Dithionite side effects become more evident with dilute hemoglobin solutions and in the presence of a large excess of oxygen. When dithionite is added to hemoglobin solutions which have been prepared with deoxygenated buffer, in order to remove only the last traces of oxygen, control experiments show that the behavior of hemoglobin is not affected4, 5. In any case careful controls should always be carried out whenever dithionite is used. In the course of a study on human hemoglobin reacted stoichiometrically in /3-93 with p-mercuribenzoate (PMB), such controls revealed the occurrence of timedependent changes in the behavior of the PMB-treated hemoglobin due to the presence of dithionite, although air was rigorously excluded from the solutions. The changes appeared in both properties of PMB-treated hemoglobin we were studying: the kinetics of combination with CO after flash photolysis and the molecular weight of the CO derivative as measmed by differential gel filtration. In view of the wide use of both PMB and dithionite as hemoglobin reagents, it appeared worthwhile to repolt these experiments in the present note. It should be recalled that upon treatment of human hemoglobin with PMB only two SH groups (those at/3-93 ) react readily. The presence of PMB on/3-93 enhances the dissociation of the protein into dimers; on the other hand, the subsequent reaction of PMB with the other SH gloups (those at a-Io 4 and/3-112) promotes further dissociation into single a and/3 chainsT, 1°. H u m a n hemoglobin was prepared from freshly drawn blood b y the (NH4)2SO 4 method and was freed from organic and inorganic ions by passage through an ionexchange resin. Pzotein concentration was calculated on the basis of an extinction coefficient E ~ = 8.4 at 54 ° n m for the CO derivative. PMB, obtained commercially, Abbreviation : PMB, p-mercuribenzoate.
Biochim. Biophys. Acta, 214 (197 o) 553-555