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A spin label for tyrosine residues
DO BBA
SHORT COMMUNICAT
33183
A spin label for tyrosine residues The technique of spin-labelling has been used to provide information about the structure and function of proteins 1-4, lipid-protein complexes 5 and membranes ". The future exploitation of the technique depends partly on the development of new labels with a defined specificity in their reaction with amino acid residues. In this communication we describe the reagent N-(2,2,5,5-tetramethyl-3-carbonyl-pyrroline-ioxyl)-imidazole (I). This reagent is an analogue of the tyrosine-specific N-acetylimidazole introduced by RIORDAN el al. 7, and has also been prepared by HAMILTONs. N
I
2,2,5,5-Tetramethylpyrroline-I-oxyl-3-carboxylic acid was prepared by the method of ROZANTZEVAND KRINITZKAYA°. N,N'-Carbonyldiimidazole was purchased from Fluka A.G., Basle. Poly-t-tyrosine (mol.wt. = IiO ooo) was purchased from Sigma. ESR spectra were obtained at 9.5 kMHz using a Varian V-45o2 X-band spectrometer. Reorientational correlation times (re) for the spin label were calculated using the theory of KIVELSON1°.
N-(2,2,5,5-Tetramethyl-3-carbonylpyrroline-z-oxyl)-imidazole.
2,2,5,5-Tetra-
methylpyrroline-I-oxyl-3-carboxylic acid (IOO mg) and N,N'-carbonyldiimidazole (2oo mg) were mixed in dry, redistilled acetone (2o ml) according to STAAB'Su method. After standing for 3o min, the solution was evaporated to dryness. The residue was dissolved in ether (5o ml) and extracted with water (5o ml) and satd. NaHCO a solution (5° ml). After drying over MgSO, and evaporation under reduced pressure, crystallisation of the residue from light petroleum (b.p. 4o-6o °) gave yellow crystals (81 mg, 66% of theory), m.p. 82-85 °. Infrared spectroscopy showed a strong carbonyl band at 171o cm -1 (KBr disc). Accurate elementary analysis was not possible due to instability of the product. Accurate mass measurement by mass spectrometry gave M = 234.1245. C12Hl~N202 requires M -- 234.1242. Spin-labelled poly-g-tyrosine. Poly-L-tyrosine (8.15 mg) was dissolved in o.I M NaOH solution (I ml). 6 mM sodium phosphate buffer (pH 7.4; 4 ml) was added and the whole solution was dialysed for 24 h against 6 mM sodium phosphate buffer (pH 7.4; 4 × 25 ° ml). To the suspension of poly-L-tyrosine (5 ml) was added spin label (I rag). The mixture was stirred for 3 h at o ° and then dialysed against 6 mM sodium phosphate (pH 7.4; 4 × 250 ml) at 4 ° for 24 h. For the pH experiment, the preparation was dialysed further for 24 h against distilled and deionised water (4 × 250 ml). t3iochim. Biophys. Acta, 194 (1969) 6 0 0 - 6 0 2
[ORT COMMUNICATIONS
¢m 10(>
80,
40 2O -(C .
fi
2
3
" "
-'2 - - . 4 5 6
s -" O'O'e'''"
. . . . 7 8pl..i9
~'~0
10 11 12 13
Fig. i. ESR spectrum of spin-labelled poly--L-tyrosine at pH 7.5. Fig. 2. Graph of intensity of low-field narrow hyperfine line of spin-labelled poly-L-tyrosine against pH.
The ESR spectrum of spin-labelled poly-L-tyrosine at pH 7.5 is shown in Fig. I. The spectrum consists of a broad component (re approx.7" lO -9 sec) probably arising from spin label molecules trapped in the helical structure of the polypeptide, and a narrow component from spin label molecules tumbling freely on ~he surface of the polypeptide. The spin label molecules are almost certainly attached to the polypeptide as O-tyrosyl esters ~.
~o ~o-~
Fig. 3. ESR spectrum of spin-labelled poly-L-tyrosine at (a) pH 11.35 and (b) pH II.6.
Biochim. Biophys. Acta, 194 (1969) 600-602
02
SHORT COMMUNICAT
The effect of pH on the E S R spectrum of spin-labelled poly-L-tyrosme is shown in Fig. 2. The intensity of the low-field narrow hyperfine line is plotted against pH under fixed spectroscopic conditions. The ESR spectra of poly-L-tyrosine at p H I 1.35 and p H I I .6 are shown in Vigs. 3a and 3b, respectively. The change in the ESR spectrum at p H 11. 5 is presumably due to a conformational change in the polypeptide structure from a-helix to random coil as the tyrosyl residues become negatively charged. FASMAN et al. 12 have observed a sharp change in tile optical rotatory dispersion spectrum of poly-L-tyr()sine at p H 11.5, the pKa of the polypeptide. At pH values in excess of 11. 5, the spin label was found to be hydrolysed from the poly-L-tyrosine at an appreciable rate. The changes in the ESR spectrum were however found to be at least partially reversible on titrating back to pH 7. Treatment of an equimolar mixture of poly-L-tyrosine and poly-L-lysine resulted in preferential labelling of tyrosine residues, suggesting that the specificity of the label for tyrosine residues is very similar to that reported for N-acetylimidazole 7. The authors wish to thank Dr. A. Morrison for carrying out the accurate mass measurement and Dr. V. B. K a m a t and Dr. A. P. Davis for stimulating discussions.
Unilever Research Laboratory, Colworth/Welwyn, The Frvthe, Welwyn, Herts. (Great Britain)
M. 1). BARRATT G . H . DODD D . CHAPMAN
1 T. J. STONE, T. BUCKMAN, P. L. NORDIO AND H. M. McCONNELL, Proc. Natl. ,lcad. Sci. U.S., 54 (1965) IOLO. 2 L. STRYER AND O. H. GRIFFIWH, Proc. Natl. Acad. Sci. U.S., 54 (1965) z785. 3 L. J. BtgRLINER AND H. M. McCONNELL, Proc. Natl. Acad. Sci. U.S., 55 (I966) 708. 4 S. OGAWA AND H. M. ~V[cCONNELL, Proc. Natl. Acad. Sci. U.S., 58 (i967) 19. 5 M. D. BARRATT, D. K. GREEN AND D. CHAPMAN, Biochim. Biophys. Acta, 152 (1968) 20. 6 D. CHAPMAN, M. D. BARRATT AND V. B. t{AMAT, Biochim. Biophys. Acta, 173 (1969) 154. 7 J. F. RIORDAN, W. E. WACKER AND B. VALLEE, Biochemistry, 4 (1965) 1758. 8 C. L. HAMILTON, u n p u b l i s h e d observations, in A. RICH AND N. DAVIDSON, Structural Chemistry and Molecular Biology, F r e e m a n , San Francisco, 1968, p. 131. 9 E. G. ROZANTZEV AND L. A. KRINITZKAYA, Tetrahedron, 2o (1964)-131. IO D. KIVELSON, J. Chem. Phys., 33 (196o) lO94. i i H. A. STAAB, Berichte, 95 (1962) 1275. 12 G. D. BASHAN, E. BODENHEIMER AND C. LINDBLOW, Biochemistry, 3 (1964) 1665.
Received August 26th, 1969 Biochim. Biophys. Acta, 194 (1969) 600-602
BBA
33179
A method for studying binding proteins, based upon differential dissociation of small ligand In plasma and certain tissue extracts, transport and receptor proteins are present which bind "specifically" and strongly small molecules like steroid hormones, with an intrinsic association constant K of the order of I • lO8-1 • lO l° M -1. However, in these biological samples, there are also other proteins and macromolecules binding Biochim. Biophys. Acta, i94 (1969) 602-605
SHORT COMMUNICAT
33183
A spin label for tyrosine residues The technique of spin-labelling has been used to provide information about the structure and function of proteins 1-4, lipid-protein complexes 5 and membranes ". The future exploitation of the technique depends partly on the development of new labels with a defined specificity in their reaction with amino acid residues. In this communication we describe the reagent N-(2,2,5,5-tetramethyl-3-carbonyl-pyrroline-ioxyl)-imidazole (I). This reagent is an analogue of the tyrosine-specific N-acetylimidazole introduced by RIORDAN el al. 7, and has also been prepared by HAMILTONs. N
I
2,2,5,5-Tetramethylpyrroline-I-oxyl-3-carboxylic acid was prepared by the method of ROZANTZEVAND KRINITZKAYA°. N,N'-Carbonyldiimidazole was purchased from Fluka A.G., Basle. Poly-t-tyrosine (mol.wt. = IiO ooo) was purchased from Sigma. ESR spectra were obtained at 9.5 kMHz using a Varian V-45o2 X-band spectrometer. Reorientational correlation times (re) for the spin label were calculated using the theory of KIVELSON1°.
N-(2,2,5,5-Tetramethyl-3-carbonylpyrroline-z-oxyl)-imidazole.
2,2,5,5-Tetra-
methylpyrroline-I-oxyl-3-carboxylic acid (IOO mg) and N,N'-carbonyldiimidazole (2oo mg) were mixed in dry, redistilled acetone (2o ml) according to STAAB'Su method. After standing for 3o min, the solution was evaporated to dryness. The residue was dissolved in ether (5o ml) and extracted with water (5o ml) and satd. NaHCO a solution (5° ml). After drying over MgSO, and evaporation under reduced pressure, crystallisation of the residue from light petroleum (b.p. 4o-6o °) gave yellow crystals (81 mg, 66% of theory), m.p. 82-85 °. Infrared spectroscopy showed a strong carbonyl band at 171o cm -1 (KBr disc). Accurate elementary analysis was not possible due to instability of the product. Accurate mass measurement by mass spectrometry gave M = 234.1245. C12Hl~N202 requires M -- 234.1242. Spin-labelled poly-g-tyrosine. Poly-L-tyrosine (8.15 mg) was dissolved in o.I M NaOH solution (I ml). 6 mM sodium phosphate buffer (pH 7.4; 4 ml) was added and the whole solution was dialysed for 24 h against 6 mM sodium phosphate buffer (pH 7.4; 4 × 25 ° ml). To the suspension of poly-L-tyrosine (5 ml) was added spin label (I rag). The mixture was stirred for 3 h at o ° and then dialysed against 6 mM sodium phosphate (pH 7.4; 4 × 250 ml) at 4 ° for 24 h. For the pH experiment, the preparation was dialysed further for 24 h against distilled and deionised water (4 × 250 ml). t3iochim. Biophys. Acta, 194 (1969) 6 0 0 - 6 0 2
[ORT COMMUNICATIONS
¢m 10(>
80,
40 2O -(C .
fi
2
3
" "
-'2 - - . 4 5 6
s -" O'O'e'''"
. . . . 7 8pl..i9
~'~0
10 11 12 13
Fig. i. ESR spectrum of spin-labelled poly--L-tyrosine at pH 7.5. Fig. 2. Graph of intensity of low-field narrow hyperfine line of spin-labelled poly-L-tyrosine against pH.
The ESR spectrum of spin-labelled poly-L-tyrosine at pH 7.5 is shown in Fig. I. The spectrum consists of a broad component (re approx.7" lO -9 sec) probably arising from spin label molecules trapped in the helical structure of the polypeptide, and a narrow component from spin label molecules tumbling freely on ~he surface of the polypeptide. The spin label molecules are almost certainly attached to the polypeptide as O-tyrosyl esters ~.
~o ~o-~
Fig. 3. ESR spectrum of spin-labelled poly-L-tyrosine at (a) pH 11.35 and (b) pH II.6.
Biochim. Biophys. Acta, 194 (1969) 600-602
02
SHORT COMMUNICAT
The effect of pH on the E S R spectrum of spin-labelled poly-L-tyrosme is shown in Fig. 2. The intensity of the low-field narrow hyperfine line is plotted against pH under fixed spectroscopic conditions. The ESR spectra of poly-L-tyrosine at p H I 1.35 and p H I I .6 are shown in Vigs. 3a and 3b, respectively. The change in the ESR spectrum at p H 11. 5 is presumably due to a conformational change in the polypeptide structure from a-helix to random coil as the tyrosyl residues become negatively charged. FASMAN et al. 12 have observed a sharp change in tile optical rotatory dispersion spectrum of poly-L-tyr()sine at p H 11.5, the pKa of the polypeptide. At pH values in excess of 11. 5, the spin label was found to be hydrolysed from the poly-L-tyrosine at an appreciable rate. The changes in the ESR spectrum were however found to be at least partially reversible on titrating back to pH 7. Treatment of an equimolar mixture of poly-L-tyrosine and poly-L-lysine resulted in preferential labelling of tyrosine residues, suggesting that the specificity of the label for tyrosine residues is very similar to that reported for N-acetylimidazole 7. The authors wish to thank Dr. A. Morrison for carrying out the accurate mass measurement and Dr. V. B. K a m a t and Dr. A. P. Davis for stimulating discussions.
Unilever Research Laboratory, Colworth/Welwyn, The Frvthe, Welwyn, Herts. (Great Britain)
M. 1). BARRATT G . H . DODD D . CHAPMAN
1 T. J. STONE, T. BUCKMAN, P. L. NORDIO AND H. M. McCONNELL, Proc. Natl. ,lcad. Sci. U.S., 54 (1965) IOLO. 2 L. STRYER AND O. H. GRIFFIWH, Proc. Natl. Acad. Sci. U.S., 54 (1965) z785. 3 L. J. BtgRLINER AND H. M. McCONNELL, Proc. Natl. Acad. Sci. U.S., 55 (I966) 708. 4 S. OGAWA AND H. M. ~V[cCONNELL, Proc. Natl. Acad. Sci. U.S., 58 (i967) 19. 5 M. D. BARRATT, D. K. GREEN AND D. CHAPMAN, Biochim. Biophys. Acta, 152 (1968) 20. 6 D. CHAPMAN, M. D. BARRATT AND V. B. t{AMAT, Biochim. Biophys. Acta, 173 (1969) 154. 7 J. F. RIORDAN, W. E. WACKER AND B. VALLEE, Biochemistry, 4 (1965) 1758. 8 C. L. HAMILTON, u n p u b l i s h e d observations, in A. RICH AND N. DAVIDSON, Structural Chemistry and Molecular Biology, F r e e m a n , San Francisco, 1968, p. 131. 9 E. G. ROZANTZEV AND L. A. KRINITZKAYA, Tetrahedron, 2o (1964)-131. IO D. KIVELSON, J. Chem. Phys., 33 (196o) lO94. i i H. A. STAAB, Berichte, 95 (1962) 1275. 12 G. D. BASHAN, E. BODENHEIMER AND C. LINDBLOW, Biochemistry, 3 (1964) 1665.
Received August 26th, 1969 Biochim. Biophys. Acta, 194 (1969) 600-602
BBA
33179
A method for studying binding proteins, based upon differential dissociation of small ligand In plasma and certain tissue extracts, transport and receptor proteins are present which bind "specifically" and strongly small molecules like steroid hormones, with an intrinsic association constant K of the order of I • lO8-1 • lO l° M -1. However, in these biological samples, there are also other proteins and macromolecules binding Biochim. Biophys. Acta, i94 (1969) 602-605