Biochimica et Biophysica Acta, 1037 (1990) 129-132
129
Elsevier BBAPRO 33539
Determination of cysteine on low-density lipoproteins using the fluorescent probe, 5-iodoacetamidofluoresceine Richard D. Coleman, Tae W. Kim, Antonio M. Gotto Jr. and Chao-yuh Yang Department of Medicine, Baylor Collegeof Medicine and The Methodist Hospital, Houston, TX (U.S.A.)
(Received20 April 1989) (Revised manuscript received5 September1989)
Key words: ApolipoproteinB-100; Amino acid sequence; Sulfliydrylpeptide; Low density lipoproteins; Lipoprotein(a); Fluorescent labeling
Using the fluorescent sulfhydryl probe, 5-iodoacetamidofluoresceine, to label the free sulfhydryl of low-density lipoprotein, the positions of two cysteine residues in apolipoprotein B were located. The tryptic peptides containing the fluorescent probe were isolated by high-performance liquid chromatography systems and sequenced by automatic techniques. The free cysteine residues of apolipoprotein B-100 on low-density lipoprotein are located at positions 3734 and 4190, either or both of which can potentially form a disulfide linkage with apolipoprotein(a) in lipoprotein(a).
Introduction
Materials and Methods
Apo B-100, the major protein component of LDL, contains a peptide ligand that binds to the L D L receptor [1]. It is an important determinant that regulates L D L metabolism. Elevated LDL-apo B plasma levels are strongly associated with increased risk of coronary artery disease. Several laboratories have recently determined the complete amino acid sequence of apo B-100 from c D N A [2-5] and its proteolytic peptides [6,7]. It is one of the largest monomeric proteins known, with a calculated molecular mass of 513 kDa. Within the L D L particle, disulfide bonds appear to influence the stability of apo B-100, which contains 25 cysteine residues. Although it is known that apo B-100 is cross-linked to apo(a) and Lp(a) by one or more disulfide bonds [8,9], the sequence position(s) of these bond(s) between the two molecules are still not known. In this paper, we report the positions of two free cysteine residues of apo B-100 on LDL, determined by using the fluorescent probe, 5-IAF, to tag sulfhydryl groups on the peptides that contain free cysteine residues.
Materials. Acetonitrile and other solvents for highperformance liquid chromatography (HPLC) were from J.T. Becker (Philipsburg, N J). Vydac C18, Hypersil ODS and Spherisorb ODS II columns were from Phenomenex (Rancho Palos Verdes, CA). Aprotinin, sodium azide, E D T A and trifluoroacetic acid were obtained from Pierce Chemical Co. (Rockford, IL). 5-Iodoacetamidofluoresceine (5-IAF) was from Molecular Probes (Eugene, OR). T o s y l p h e n y l a l a n i n e c h l o r o m e t h a n e treated (TPCK) trypsin was from Worthington Biochemicals (Freehold, N J). Reagents and solvents for the automatic gas-phase sequencer were the products of Applied Biosystems (Foster City, CA). Preparation of L D L . Blood was drawn from a normolipemic nonfasting donor (male, 31 y) at The Methodist Hospital blood center. Aprotinin (0.055 units/ml), sodium azide (5.00 u g / m l ) and EDTA (5.08 u g / m l ) were added to the plasma. L D L (density 1.025-1.055 g / m l ) was purified using a KBr gradient and dialyzed against 0.100 M Tris-HC1 (pH 8.5). The protein was analyzed by electrophoresis on 1% S D S / 5 - 1 4 % gradient polyacrylamide slab gel and was shown to contain only one protein band at the position of apo B-100. Derioatization o f L D L . 5-IAF was dissolved in 0.100 M Tris-HC1 (pH 8.5) and added to the LDL (100-fold molar excess of 5-IAF). The mixture was stirred in the dark at room temperature for 16 h. To eliminate excess reagent, the sample was applied to a 3 × 20 cm Sephadex G-25 column and eluted with 0.100 M ammonium
Abbreviations: 5-IAF, 5-iodoacetamidofluoresceine;apo, apolipoprotein; HPLC, high-performance liquid chromatography; LDL, low-density lipoprotein; Lp(a), lipoprotein(a); TFA, trifluoroacetic
acid. Correspondence: C.-Y. Yang, Department of Medicine, Baylor College of Medicine, The Methodist Hospital, 6565 Farmin, MS A-601, Houston, TX 77030, U.S.A.
0167-4838/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
130 bicarbonate (pH 8.0). The fractions containing fluorescent material eluting in the void volume were pooled and lyophilized. The derivatized L D L was delipidated by extracting four times with 40 ml of ether/ethanol (3:1). Each extraction was carried out by adding the solvent to the sample, vortexing, placing the sample in a freezer ( - 20 ° C) for 1 h, centrifuging at low speed, and removing the solvent by aspiration. Tryptic digestion. 100 mg of delipidated apo B-100 was resuspended in 15 ml of 0.100 M ammonium bicarbonate (pH 8.0) and 2 mg of TPCK-treated trypsin was added to initiate digestion overnight. Isolation of the peptides. The tryptic peptides were separated by HPLC using three different conditions [10]. First, the sample was applied to a Vydac C 4 6.25 × 250 mm reverse-phase column and eluted with a linear gradient of 1% per min, using a trifluoroacetic acid buffer system (A: 0.1% TFA in water; B: 0.08% TFA in 95% acetonitrile and 5% water). Second, each of the fluorescent peaks was collected and rechromatographed with a Phenomenex 4.6 x 250 ram, 5 /xm Hypersil ODS column using a phosphate buffer system. Buffer A consisted of 5 mM sodium phosphate (pH 6.0) and buffer B was 90% acetonitrile and 10% water. A third separation was carried out using the same column and buffers as in the second separation, except that the pH of buffer A was adjusted to 4.0. All the separations were performed at 50 ° C; UV and fluorescence detectors from Applied Biosystems were used. Characterization of the peptides. The fluorescent peptides were sequenced with an Applied Biosystems automated gas-phase sequencer. The amino acid compositions of the peptides were determined after acid hydrolysis followed by conversion of the amino acids to their phenylthiocarbamyl derivatives as previously described [11]. The analyses were carried out using a Hewlett-Packard 1090M HPLC system and a 4.6 x 150 mm Phenomenex 3 /~m Spherisorb ODS II column as described previously [12]. Results and Discussion
To determine the location of the free cysteine residues of apo B-100 in LDL, it was desirable to use a reagent that was easily detectable while also being soluble in an aqueous buffer of p H 8.5. 5-iodoacetamidofluoresceine met these requirements. The reagent's high absorption coefficient (75000 cm -1 • M -1 at pH 8.5 and 493 nm), high quantum efficiency [13], and favorable emission wavelength (550 nm) make it a very sensitive probe. Since the absorption spectrum of 5-IAF varies with pH, an optimal excitation wavelength was selected for the pH of each individual buffer. Fig. 1 shows the first HPLC separation of the tryptic peptides of apo B-100 labeled with 5-IAF. Two systems were used to detect all peptides and also specifically
art
c~
50
0
10
20
30
40
i'
50
Time (rnin.)
Fig. 1. Primary purification of the tryptic peptides of 5-IAF labeled apo B-100. Tryptic peptides were separated on a Vydac C a column (6.25 × 250 m m ) using a T F A buffer system. The fluorescence excitation wavelength was 436 n m and the emission cutoff was 470 nm.
identify fluorescently labeled fragments. The first system measured absorbance at 220 nm, while the second measured fluorescence, using an excitation wavelength of 493 nm and emission above 550 nm. All fluorescent fractions, including major peaks 1-5 and minor peaks A - H , were collected and rechromatographed with a Hyperils ODS column and phosphate buffer system. The purified fluorescent fractions were then subjected to N-terminal analysis to check purity. Pure fluorescent peptides were sequenced and analyzed for amino acid composition; impure peptides were rechromatographed with the same buffer system, but with the pH lowered from 6 to 4. Our results show that peaks 1, 2, A, B, C and D were not associated with any peptides that contain a Cys residue. Peaks 3, 4 and E were associated with a peptide that contained a Cys residue at position 4190 of the apo B-100. The peaks 5, F, G and H contained a peptide with Cys3734. Peptide 1 (position 3710-3735) was isolated from peaks 5, G and H, and peptide 2 (position 4187-4185) was from peaks 3 and 4. As an example of how fluorescent fractions were purified, Fig. 2 shows the rechromatography of peak 5. Each of the three major fluorescence peaks contained peptide 1. Peak 5-3 in this chromatogram was collected and subjected to a third HPLC separation. As shown in the Fig. 2 insets, peaks 5-3-1 and 5-3-2 were finally isolated in a pure form (Tables I and II). Peptide 1 was obtained after two HPLC purifications. With the same procedure, peptide 2 was isolated. The amino acid compositions of the two different peptides that were found confirmed their identity and length (Table I). The specific position of each amino acid residue verified by sequence analysis is underlined and shown in Table II. The fluorescently-labeled cysteine residues did not appear in the HPLC chromato-
131 I 15-3
100
TABLE II The sequences of the two peptides containing free cysteine residues
Underlined residues were sequenced by gas-phase sequencing. The 5-IAF Cys did not show up in the gas-phase sequencer data and the fluorescence remained on the filtration after sequencing.
7
m . . . . ,re,o,
S l
Peptide
Residue numbers
Sequence
1 2
3710-3735 4187-4195
LNDLNSVLVHPTFHVPFTDLQVPSCK EELCTRF IR
,.
0
I 20
10
I 30
I 40
0
Tlme (min.)
Fig. 2. HPLC separation of peak 5 from Fig. 1. The separation was carried out using a linear gradient of 0 to 45% B in 45 min on a Hypersil ODS column (4.6 × 250 mm). Buffer A was 5 mM sodium phosphate (pH 6.0) and buffer B was 90% acetonitrile and 10% water. The fluorescence excitation wavelength was 493 nm and the emission cutoff was 550 nm. The insert is the chromatogram of second rechromatography of peak 5-3. The HPLC conditions were the same as before, except the pH of the buffer system was changed from 6 to 4.
gram from the gas-phase peptide sequencer a n d the fluorescence could be seen on the filter after sequencing. This indicates that the 5 - I A F labeled cysteine was n o t extracted from the filter. Lack of a n y fluorescently labeled peptides other t h a n the two m e n t i o n e d ( a m i n o acid positions 3710-3735 a n d 4187-4195) indicated that the derivatization reac-
TABLE I Amino acid analysis data for the two peptides containing free cysteine residues.
Amino
Peptide 1
Peptide 2
Acid
Expected
Found
Expected
Found
Asx Glx Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys * Ile Leu Phe Lys
4 1 2 . 1 2 3 . 4 1 1 4 2 1
5.00 1.23 2.10 . 1.04
2 -
2.01 -
1 1 -
0.206 0.844 -
1 1 1 1 1 -
1.56 1.04 1.04 1.30 -
.
.
1.85
.
3.16 . 3.19 0.949 4.33 2.14 0.988
.
* The analysis did not quantitate cysteine.
tion was highly specific. T h e presence of multiple peaks from a single p e p t i d e m a y result from ion pairing with the buffer salts or the peptides m a y exist in multiple c o n f o r m a t i o n s with differing c h r o m a t o g r a p h i c properties. T h e overall yields of the peptides are approx. 45%. T h e use of the gel-filtration step before delipidation greatly facilitated isolation of the labeled peptides. O m i s s i o n of this step resulted in a variety of very large fluorescence peaks which p r o h i b i t e d the d e t e r m i n a t i o n of which peaks were actually peptides. Even after gel filtration, two very large fluorescent peaks r e m a i n e d in the first H P L C s e p a r a t i o n (Fig. 1), b u t after the final c h r o m a t o g r a p h y step, b o t h gas-phase sequencing a n d a m i n o acid analysis showed that this fluorescent material was n o t a peptide. Analysis of small peaks of fluorescent material ( A - H ) eluted from the Vydac C a (Fig. 1) revealed that n o further sulfhydryl peptides other t h a n Cys-3734 a n d Cys-419o were found. Peaks A to D gave the same negative results as peaks 1 a n d 2, which c o n t a i n e d n o peptides. Peaks E a n d F c o n t a i n e d the c h y m o t r y p t i c side reaction product. Both peptides were cleaved after p h e n y l a l a n i n e . It is possible that apo B-100 m a y c o n t a i n more then two free cysteine residues; however, d u e to its smaller accessibility to the probe, only Cys-3734 a n d Cys-4190 were identified b y these procedures. A p o B-100 c o n t a i n s 25 cysteine residues. The inform a t i o n a b o u t the disulfide linkages a n d the free sulfhydryl groups of apo B-100 is still limited. H u a n g et al. [14] reported that apo B-100 might c o n t a i n thioester linkages. We have used a very sensitive sulfhydryl p r o b e to locate the p o s i t i o n of two free cysteine residues o n LDL. These are located at the C - t e r m i n a l e n d of the p r o t e i n (Cys-3734, Cys-4190)"W h e t h e r o n e or b o t h of the free residues are involved i n disulfide linkage with apo(a) or lp(a) will be the subject of future investigations. Acknowledgements
W e t h a n k B. T o u c h s t o n e a n d M.L. Y a n g for excellent technical assistance, S. Kelly for the artwork, a n d M. N e e d h a m for p r e p a r a t i o n of this m a n u s c r i p t . This work was s u p p o r t e d b y a g r a n t from the N a t i o n a l Institutes of H e a l t h ( N a t i o n a l Research a n d D e m o n stration C e n t e r i n Arteriosclerosis, HL-27341), a grant-
132 i n - a i d f r o m the A m e r i c a n H e a r t A s s o c i a t i o n ( N o . 870863), a n d a g r a n t f r o m T h e M e t h o d i s t H o s p i t a l .
References 1 Brown, M.S. and Goldstein, J.L. (1986) Science 232, 34-47. 2 Chen, S.-H., Yang, C.Y., Chela, P.-F. Setzer, D., Tanimura, M., Li, W.-H., Gotto, A.M., Jr. and Chan, L. (1986) J. Biol. Chem. 261, 12918-12921. 3 Knott, T. J., Wallis, S.C., Powell, L.M., Pease, R.J., Lusis, A. J., Blackhart, B., McCarthy, B.J., Mahley, R.W., Levy-Wilson, B. and Scott, J. (1986) Nucl. Acids Res. 14, 7501-7503. 4 Law, S.W., Grant, S.M., Higuchi, K., Hospattankar, A., Lackner, K., Lee, N. and Brewer, H. B., Jr. (1986) Proc. Natl. Acad. Sci. USA 83, 8142-8146. 5 Cladaras, C., Hadzopoulou-Cladaras, M., Nolte, R.T., Atkinson, D. and Zannis, V.I. (1986) EMBO J. 5, 3495-3502. 6 Yang, C.Y., Chen, S.H., Gianturco, S.A., Bradley, W.B., Sparrow,
7
8 9 10 11 12 13 14
J.T., Tanimura, M., Li, W. H., Sparrow, D. A., DeLoof, H., Rosseneu, M., Lee, F.S., Gu, Z.W., Gotto, A.M., Jr. and Chan, L. (1986) Nature 323, 738-742. Yang, C.Y., Gu, Z.W., Weng, S.A., Kim, T.W., Chen, S.H., Pownall, H.J., Sharp, P.M., Liu, S.W., Li, W.H., Gotto, A.M. Jr. and Chan, L. (1989) Arteriosclerosis 9, 96-108. Utermann, G. and Weber, W. (1983) FEBS Lett. 154, 357-361. Gaubatz, J.W., Heideman, C., Gotto, A.M., Morrisett, J.D. and Dahlen, G.H. (1983) J. Biol. Chem. 258, 4582-4589. Yang, C.Y., Yang, T., Pownall, H.J. and Gotto, A.M., Jr. (1986) Eur. J. Biochem. 160, 427-431. Heinrikson, R.L. and Meredith, S.C. (1984) Anal. Biochem. 136, 65-74. Yang, C.Y. and Sepulveda, F.I. (1985) J. Chromatogr. 346, 413-416. Hirsh, R.E., Zukin, R.S. and Nagel, R.L. (1986) Biochem. Biophys. Res. Commun. 138, 489-493. Huang, G., Lee, D.M. and Singh S. (1988) Biochemistry 27, 1395-1400.
129
Elsevier BBAPRO 33539
Determination of cysteine on low-density lipoproteins using the fluorescent probe, 5-iodoacetamidofluoresceine Richard D. Coleman, Tae W. Kim, Antonio M. Gotto Jr. and Chao-yuh Yang Department of Medicine, Baylor Collegeof Medicine and The Methodist Hospital, Houston, TX (U.S.A.)
(Received20 April 1989) (Revised manuscript received5 September1989)
Key words: ApolipoproteinB-100; Amino acid sequence; Sulfliydrylpeptide; Low density lipoproteins; Lipoprotein(a); Fluorescent labeling
Using the fluorescent sulfhydryl probe, 5-iodoacetamidofluoresceine, to label the free sulfhydryl of low-density lipoprotein, the positions of two cysteine residues in apolipoprotein B were located. The tryptic peptides containing the fluorescent probe were isolated by high-performance liquid chromatography systems and sequenced by automatic techniques. The free cysteine residues of apolipoprotein B-100 on low-density lipoprotein are located at positions 3734 and 4190, either or both of which can potentially form a disulfide linkage with apolipoprotein(a) in lipoprotein(a).
Introduction
Materials and Methods
Apo B-100, the major protein component of LDL, contains a peptide ligand that binds to the L D L receptor [1]. It is an important determinant that regulates L D L metabolism. Elevated LDL-apo B plasma levels are strongly associated with increased risk of coronary artery disease. Several laboratories have recently determined the complete amino acid sequence of apo B-100 from c D N A [2-5] and its proteolytic peptides [6,7]. It is one of the largest monomeric proteins known, with a calculated molecular mass of 513 kDa. Within the L D L particle, disulfide bonds appear to influence the stability of apo B-100, which contains 25 cysteine residues. Although it is known that apo B-100 is cross-linked to apo(a) and Lp(a) by one or more disulfide bonds [8,9], the sequence position(s) of these bond(s) between the two molecules are still not known. In this paper, we report the positions of two free cysteine residues of apo B-100 on LDL, determined by using the fluorescent probe, 5-IAF, to tag sulfhydryl groups on the peptides that contain free cysteine residues.
Materials. Acetonitrile and other solvents for highperformance liquid chromatography (HPLC) were from J.T. Becker (Philipsburg, N J). Vydac C18, Hypersil ODS and Spherisorb ODS II columns were from Phenomenex (Rancho Palos Verdes, CA). Aprotinin, sodium azide, E D T A and trifluoroacetic acid were obtained from Pierce Chemical Co. (Rockford, IL). 5-Iodoacetamidofluoresceine (5-IAF) was from Molecular Probes (Eugene, OR). T o s y l p h e n y l a l a n i n e c h l o r o m e t h a n e treated (TPCK) trypsin was from Worthington Biochemicals (Freehold, N J). Reagents and solvents for the automatic gas-phase sequencer were the products of Applied Biosystems (Foster City, CA). Preparation of L D L . Blood was drawn from a normolipemic nonfasting donor (male, 31 y) at The Methodist Hospital blood center. Aprotinin (0.055 units/ml), sodium azide (5.00 u g / m l ) and EDTA (5.08 u g / m l ) were added to the plasma. L D L (density 1.025-1.055 g / m l ) was purified using a KBr gradient and dialyzed against 0.100 M Tris-HC1 (pH 8.5). The protein was analyzed by electrophoresis on 1% S D S / 5 - 1 4 % gradient polyacrylamide slab gel and was shown to contain only one protein band at the position of apo B-100. Derioatization o f L D L . 5-IAF was dissolved in 0.100 M Tris-HC1 (pH 8.5) and added to the LDL (100-fold molar excess of 5-IAF). The mixture was stirred in the dark at room temperature for 16 h. To eliminate excess reagent, the sample was applied to a 3 × 20 cm Sephadex G-25 column and eluted with 0.100 M ammonium
Abbreviations: 5-IAF, 5-iodoacetamidofluoresceine;apo, apolipoprotein; HPLC, high-performance liquid chromatography; LDL, low-density lipoprotein; Lp(a), lipoprotein(a); TFA, trifluoroacetic
acid. Correspondence: C.-Y. Yang, Department of Medicine, Baylor College of Medicine, The Methodist Hospital, 6565 Farmin, MS A-601, Houston, TX 77030, U.S.A.
0167-4838/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
130 bicarbonate (pH 8.0). The fractions containing fluorescent material eluting in the void volume were pooled and lyophilized. The derivatized L D L was delipidated by extracting four times with 40 ml of ether/ethanol (3:1). Each extraction was carried out by adding the solvent to the sample, vortexing, placing the sample in a freezer ( - 20 ° C) for 1 h, centrifuging at low speed, and removing the solvent by aspiration. Tryptic digestion. 100 mg of delipidated apo B-100 was resuspended in 15 ml of 0.100 M ammonium bicarbonate (pH 8.0) and 2 mg of TPCK-treated trypsin was added to initiate digestion overnight. Isolation of the peptides. The tryptic peptides were separated by HPLC using three different conditions [10]. First, the sample was applied to a Vydac C 4 6.25 × 250 mm reverse-phase column and eluted with a linear gradient of 1% per min, using a trifluoroacetic acid buffer system (A: 0.1% TFA in water; B: 0.08% TFA in 95% acetonitrile and 5% water). Second, each of the fluorescent peaks was collected and rechromatographed with a Phenomenex 4.6 x 250 ram, 5 /xm Hypersil ODS column using a phosphate buffer system. Buffer A consisted of 5 mM sodium phosphate (pH 6.0) and buffer B was 90% acetonitrile and 10% water. A third separation was carried out using the same column and buffers as in the second separation, except that the pH of buffer A was adjusted to 4.0. All the separations were performed at 50 ° C; UV and fluorescence detectors from Applied Biosystems were used. Characterization of the peptides. The fluorescent peptides were sequenced with an Applied Biosystems automated gas-phase sequencer. The amino acid compositions of the peptides were determined after acid hydrolysis followed by conversion of the amino acids to their phenylthiocarbamyl derivatives as previously described [11]. The analyses were carried out using a Hewlett-Packard 1090M HPLC system and a 4.6 x 150 mm Phenomenex 3 /~m Spherisorb ODS II column as described previously [12]. Results and Discussion
To determine the location of the free cysteine residues of apo B-100 in LDL, it was desirable to use a reagent that was easily detectable while also being soluble in an aqueous buffer of p H 8.5. 5-iodoacetamidofluoresceine met these requirements. The reagent's high absorption coefficient (75000 cm -1 • M -1 at pH 8.5 and 493 nm), high quantum efficiency [13], and favorable emission wavelength (550 nm) make it a very sensitive probe. Since the absorption spectrum of 5-IAF varies with pH, an optimal excitation wavelength was selected for the pH of each individual buffer. Fig. 1 shows the first HPLC separation of the tryptic peptides of apo B-100 labeled with 5-IAF. Two systems were used to detect all peptides and also specifically
art
c~
50
0
10
20
30
40
i'
50
Time (rnin.)
Fig. 1. Primary purification of the tryptic peptides of 5-IAF labeled apo B-100. Tryptic peptides were separated on a Vydac C a column (6.25 × 250 m m ) using a T F A buffer system. The fluorescence excitation wavelength was 436 n m and the emission cutoff was 470 nm.
identify fluorescently labeled fragments. The first system measured absorbance at 220 nm, while the second measured fluorescence, using an excitation wavelength of 493 nm and emission above 550 nm. All fluorescent fractions, including major peaks 1-5 and minor peaks A - H , were collected and rechromatographed with a Hyperils ODS column and phosphate buffer system. The purified fluorescent fractions were then subjected to N-terminal analysis to check purity. Pure fluorescent peptides were sequenced and analyzed for amino acid composition; impure peptides were rechromatographed with the same buffer system, but with the pH lowered from 6 to 4. Our results show that peaks 1, 2, A, B, C and D were not associated with any peptides that contain a Cys residue. Peaks 3, 4 and E were associated with a peptide that contained a Cys residue at position 4190 of the apo B-100. The peaks 5, F, G and H contained a peptide with Cys3734. Peptide 1 (position 3710-3735) was isolated from peaks 5, G and H, and peptide 2 (position 4187-4185) was from peaks 3 and 4. As an example of how fluorescent fractions were purified, Fig. 2 shows the rechromatography of peak 5. Each of the three major fluorescence peaks contained peptide 1. Peak 5-3 in this chromatogram was collected and subjected to a third HPLC separation. As shown in the Fig. 2 insets, peaks 5-3-1 and 5-3-2 were finally isolated in a pure form (Tables I and II). Peptide 1 was obtained after two HPLC purifications. With the same procedure, peptide 2 was isolated. The amino acid compositions of the two different peptides that were found confirmed their identity and length (Table I). The specific position of each amino acid residue verified by sequence analysis is underlined and shown in Table II. The fluorescently-labeled cysteine residues did not appear in the HPLC chromato-
131 I 15-3
100
TABLE II The sequences of the two peptides containing free cysteine residues
Underlined residues were sequenced by gas-phase sequencing. The 5-IAF Cys did not show up in the gas-phase sequencer data and the fluorescence remained on the filtration after sequencing.
7
m . . . . ,re,o,
S l
Peptide
Residue numbers
Sequence
1 2
3710-3735 4187-4195
LNDLNSVLVHPTFHVPFTDLQVPSCK EELCTRF IR
,.
0
I 20
10
I 30
I 40
0
Tlme (min.)
Fig. 2. HPLC separation of peak 5 from Fig. 1. The separation was carried out using a linear gradient of 0 to 45% B in 45 min on a Hypersil ODS column (4.6 × 250 mm). Buffer A was 5 mM sodium phosphate (pH 6.0) and buffer B was 90% acetonitrile and 10% water. The fluorescence excitation wavelength was 493 nm and the emission cutoff was 550 nm. The insert is the chromatogram of second rechromatography of peak 5-3. The HPLC conditions were the same as before, except the pH of the buffer system was changed from 6 to 4.
gram from the gas-phase peptide sequencer a n d the fluorescence could be seen on the filter after sequencing. This indicates that the 5 - I A F labeled cysteine was n o t extracted from the filter. Lack of a n y fluorescently labeled peptides other t h a n the two m e n t i o n e d ( a m i n o acid positions 3710-3735 a n d 4187-4195) indicated that the derivatization reac-
TABLE I Amino acid analysis data for the two peptides containing free cysteine residues.
Amino
Peptide 1
Peptide 2
Acid
Expected
Found
Expected
Found
Asx Glx Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys * Ile Leu Phe Lys
4 1 2 . 1 2 3 . 4 1 1 4 2 1
5.00 1.23 2.10 . 1.04
2 -
2.01 -
1 1 -
0.206 0.844 -
1 1 1 1 1 -
1.56 1.04 1.04 1.30 -
.
.
1.85
.
3.16 . 3.19 0.949 4.33 2.14 0.988
.
* The analysis did not quantitate cysteine.
tion was highly specific. T h e presence of multiple peaks from a single p e p t i d e m a y result from ion pairing with the buffer salts or the peptides m a y exist in multiple c o n f o r m a t i o n s with differing c h r o m a t o g r a p h i c properties. T h e overall yields of the peptides are approx. 45%. T h e use of the gel-filtration step before delipidation greatly facilitated isolation of the labeled peptides. O m i s s i o n of this step resulted in a variety of very large fluorescence peaks which p r o h i b i t e d the d e t e r m i n a t i o n of which peaks were actually peptides. Even after gel filtration, two very large fluorescent peaks r e m a i n e d in the first H P L C s e p a r a t i o n (Fig. 1), b u t after the final c h r o m a t o g r a p h y step, b o t h gas-phase sequencing a n d a m i n o acid analysis showed that this fluorescent material was n o t a peptide. Analysis of small peaks of fluorescent material ( A - H ) eluted from the Vydac C a (Fig. 1) revealed that n o further sulfhydryl peptides other t h a n Cys-3734 a n d Cys-419o were found. Peaks A to D gave the same negative results as peaks 1 a n d 2, which c o n t a i n e d n o peptides. Peaks E a n d F c o n t a i n e d the c h y m o t r y p t i c side reaction product. Both peptides were cleaved after p h e n y l a l a n i n e . It is possible that apo B-100 m a y c o n t a i n more then two free cysteine residues; however, d u e to its smaller accessibility to the probe, only Cys-3734 a n d Cys-4190 were identified b y these procedures. A p o B-100 c o n t a i n s 25 cysteine residues. The inform a t i o n a b o u t the disulfide linkages a n d the free sulfhydryl groups of apo B-100 is still limited. H u a n g et al. [14] reported that apo B-100 might c o n t a i n thioester linkages. We have used a very sensitive sulfhydryl p r o b e to locate the p o s i t i o n of two free cysteine residues o n LDL. These are located at the C - t e r m i n a l e n d of the p r o t e i n (Cys-3734, Cys-4190)"W h e t h e r o n e or b o t h of the free residues are involved i n disulfide linkage with apo(a) or lp(a) will be the subject of future investigations. Acknowledgements
W e t h a n k B. T o u c h s t o n e a n d M.L. Y a n g for excellent technical assistance, S. Kelly for the artwork, a n d M. N e e d h a m for p r e p a r a t i o n of this m a n u s c r i p t . This work was s u p p o r t e d b y a g r a n t from the N a t i o n a l Institutes of H e a l t h ( N a t i o n a l Research a n d D e m o n stration C e n t e r i n Arteriosclerosis, HL-27341), a grant-
132 i n - a i d f r o m the A m e r i c a n H e a r t A s s o c i a t i o n ( N o . 870863), a n d a g r a n t f r o m T h e M e t h o d i s t H o s p i t a l .
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