Int. J. Peptide PTotein Res. 13,1979,498-509 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

Q U A N T I T A T I O N O F SOME AMINO-TERMINAL R E S I D U E S IN PROTEINS U S I N G 3H-LABELLED D A N S Y L CHLORIDE A N D 14C-LABELLED AMINO ACIDS RAGNAR FLENGSRUD

Institute o f Medical Biology, University o f Troms4, Tromsd, Norway

Received 17 March, accepted for publication 29 September 1978 A method for quantitation o f amino-terminal residues in proteins is presented. The method is a modification o f a double isotope-labelling technique, using 3H-labelled dansyl chloride and ‘‘C-labelled amino acids as internal standards. The method is demonstrated on human fibrinogen, horse myoglobin and on mouse myoloma IgA. A linear relationship between the ratio 3H/‘4C in the separated amino-terminal amino acid of the protein and the amount of protein added in the labelling mixture was obtained with standard deviations b f f 7.4%, f 3.4% and 51 0.3%, respectively. A n application o f the method is demonstrated by measuring the increase in amino-terminal glycine in fibrinogen following the proteolytic action of thrombin. The method seems to be useful when 0.1 nmol or more of protein is used. Key words: dansyl chloride; IgA; myoglobin; quantitative amino-terminal analysis; thrombin-fibrinogen reaction.

a review, Neuhoff (1973) concluded that only semiquantitative data might be obtained. Rapoport et al. (1967) reported quantitative determination of the amino-terminal residues of insulin using I4C-labelleddansyl chloride, but this method was not applicable to proteins of higher molecular weight. In addition, Schmer (1 967) determined quantitatively the aminoterminal residues in immunoglobulins with a detection level of 0.1 nmol and a standard deviation of f 10% using the extraction method of Seiler & Weichman (1 966). The problems with the sensitivity of the dansyl reaction to the various parameters were overcome by Brown & Perham (1973) and Abbreviations used: Dansyl chloride, DanC1, 1 Snodgrass & Iversen (1973) who introduced dimethylamino-naphthaleneSsulphony1chloride; IgA, a double-isotope labelling technique (Keston immunoglobulin A; S.D., standard deviation; TCA, et al., 1950) using 3H-dansyl chloride and trichloroacetic acid.

Since the introduction of dansyl chloride (Weber, 1952; Hartley & Massey, 1956) this technique has become a valuable and widely used tool in qualitative micro-scale analysis of amino acids, peptides and proteins. However, very few reports have appeared on the use of dansyl chloride in quantitative analysis. Studies by Neadle & Pollit (1965), Gros & Labouesse (1969), Hartley (1970), Brie1 & Neuhoff (1972) and Gray (1972) showed that the reaction of dansyl chloride with amino acids, peptides and proteins depended on a number of parameters which were difficult to control. In

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0367-8377/79/0.50498-12

$02.00/00 1979 Munksgaard, Copenhagen

QUANTITATION OF AMINO-TERMINAL RESIDUES

14C-amino acids for quantitative amino acid analysis. Later, similar methods were presented (Burzynski, 1975; Joseph & Halliday, 1975). This method will correct for losses both in the dansylation procedure and in the chromatographic separation. Unfortunately, this strategy is not directly applicable to proteins unless the same protein with a 14C-labelled amino-terminal residue is used as an internal standard. Since such proteins are not easily obtained, the appropriate 14C-labelled free amino acids were added to the proteins and the mixture was allowed to react with 3H-labelled dansyl chloride in a completely volatile buffer. However, since the amino acids and proteins react with dansyl chloride at different rates (Gros & Labouesse, 1969; Gray, 1972) the internal standard cannot accurately correct for losses in the dansylation reaction. However, the losses in dansylation of proteins relative to that of the internal standard seem to be independent of the amount of protein added. The present method is demonstrated (1) on human fibrinogen, which has two each of pyroglutamic acid, alanine and tyrosine as amino-terminal residues ( b r a n d & Middlebrook, 1953; Blomback & Yamashina, 1958; Blomback et al., 1963), (2) on equine myoglobin, which has &cine as amhxAemirra1 residue (Porter & Sanger, 1948; U e s o n & Theorell, 1960) and (3) on mouse myoloma IgA-protein, which has two pyroglutamic acids and two asparagines as amino-terminal residues (Francis et al., 1974). Since the yield of the labelling of amino-terminal residues seems to vary considerably with the different amino-terminal residues, the method does not permit determination of moles of aminoterminal residues per mole of protein. It is suggested that the method might be useful for studies on the rate of specific proteolytic reaction by quantitation of newly appearing amino-terminal residues, as demonstrated on the thrombin-fibrinogen reaction. MATERIAL

Radiochemicals were from the Radiochemical Centre, Amersham, Bucks, U.K. Amino acids, bovine serum albumin (crystallized and lyophilized) and myoglobin (equine skeletal

muscle, type I) were from Sigma Chem. Co., St. Louis, Mo., U.S.A. Mouse myoloma IgA (protein 3 15) was purified according to Goetzl & Metzger (1970) and generously supplied by Gustav Gaudernack, from our institute. The protein, in 0.01 M sodium phosphate buffer, pH 7.4 and 0.1 5 M NaCl, contained 2.22 mg/ml, or 14.8 pM assuming El&%-, cm = 1.44 and a molecular weight of 150000 (Eisen et al., 1970; Rockey et al., 1971). The snake venom from Echis carinatus was a generous gift from Dr. F. Kornalik, Institute of Pathophysiology, Charles University, Prague, CSSR. Whatman cellulose@CM-32 and DE-32 was from Balstone Ltd., Maidstone, Kent, U.K. Sephadexo G-25 (medium) and G-100 were from Pharmacia, Uppsala, Sweden. Instagelo and Dilusolve@ were from Packard Instruments, A. Randmael, Oslo, Norway. Dansyl chloride and polyamide sheets were from BDH, Poole, Dorset, U.K. Polyamide sheets (F 1700) were also obtained from Schleicher & SchiiU, Dassel, W. Germany. Hyamine hydroxide (Hyamine-10-X)was from Koch-Light Lab., Colnbrook, U.K. Bovine thrombin was from Hoffman-La Roche, Basel, Switzerland. XM-100 A and UM-10 Amicon@ Filters were obtained from Amicon, Lexington, Mass., U.S.A. Test tubes (Pyrex, rimless, 10 x 75 mm) from Jobling, Sunderland, U.K. were cleaned in chromosulphuric acid and dried before use. Pyridine was purified according to Jones (1970). Double-distilled water was used in all aqueous solutions. All other reagents were of analytical grade. EXPERIMENTAL PROCEDURES AND RESULTS Preparation of the radiochemical solutions

The dansyl chloride batches were prepared and stored as described by Brown & Perham (1973) with the exception that 57.5 mg unlabelled dansyl chloride was added to lmCi of 3Hdansyl chloride and the mixture redissolved in 2.6ml acetone. Aliquots of this solution were withdrawn while the solution was kept on ice (Neuhoff, 1973). Four different 'H-dansyl chloride batches were used. In the fibrinogen and myoglobin experiments the following mixture of 14C-labelled 499

R. FLENGSRUD

amino acids was used as internal standard: lop1 each of (U-14C) L-alanine (CFB 62, 49pCi/ml, 159mCi/mmol), (U-14C) L-glycine (CFB 66, 50pCi/ml, 114mCi/mmol) and (U-14C)~tyrosine (CFB 74, 50pCi/m 1,483mCi/mmol) and the mixture was diluted to a final volume of 2.0ml with water. In the IgA experiment a 3-fold dilution in water of the L-(U-'~C)amino acid mixture (CFB 104,54mCi/mAtom carbon containing (by activity) 9% L-aspartic acid) was used. All radiochemicals were stored at -20°C. Standard solutions of amino acids An aqueous mixture consisting of L-alanine,

L-glycine and L-tyrosine and an aqueous solution of L-aspartic acid was made. The concentration of each amino acid was determined using a JEOL (JLC 6 AH) amino acid analyzer using norleucine as the internal standard. An aqueous solution of L-alanine (100pM) and ) also made. L-glycine 4100 p ~ was Drying 'of samples

Samples were dried in a vacuumchamber at room temperature in the presence of NaOH and P, 0 5 . Performic acid oxidation This was performed at 0' by the procedure of

Hirs (1956). The dried protein samples were dissolved in 50pl 99% formic acid prior to the oxidation. After the oxidation the samples were dried overnight to ensure complete drying of the samples. Purification of human thrombin

Prothrombin was purified from human plasma as described for the purification of an aminoterminal fragment of prothrombin from serum (Skotland el al., 1974) with the exception that preparative gel electrophoresis was carried out only once. Prothrombin was then activated by snake venom from Echis carinatus as follows: 245 ml prothrombin (60pg/ml in 0.05 M tris-HC1/0.1 M NaC1, pH 7.5) was mixed with 0.4 ml of the Echis carinatus reagent (1 mg/ml in 0.05M tris-HCl, pH 7.4) and incubated for 30min at 37' and the thrombin generated was precipitated by addition of solid NH4 SO4 to 75% saturation (Gorman & Castaldi, 1974). The precipitate was dissolved in 0.05 M trisc HCl/O.Ol M NaC1, pH 8.0 and dialyzed against 500

the same buffer at 4", then applied to a DEAEcellulose (DE-32) column (2 x 6 cm) previously equilibrated against the same buffer. The thrombin was then eluted by a linear gradient consisting of 160 ml of 0-0.5 M NaCl in the equilibration buffer. The fractions with thrombin activity (Flengsrud et al., 1972) were pooled and concentrated in an Amicon ultrafiltration cell with an UM-10 filter to a final volume of 24.4ml and a protein concentration of 120 pg/ml. The thrombin preparation was stored at -70". Purification of human fibrinogen

Fibrinogen (preparation I) was purified from human plasma by the method of Jakobsen & Kierulf (1973). In addition the fibrinogen obtained by this method was submitted to gel filtration on a Sephadex G-100 column (2.2 x 81 cm) equilibrated and eluted with 2.9 mM veronal-HC1 buffer, pH 7.0, containing 0.3 M NaCl. The fractions containing fibrinogen were pooled and concentrated in an Amicon cell using an XM-100 A filter. For preparations I1 and I11 the following modifications were introduced. Blood was collected with l/lOvol. anticoagulant containing 0.1 M Na-oxalate, 0.2 M benzamidine and 0.5 M e-aminocaproid acid. The resulting fibrinogen solution was transferred to an Amicon ultrafiltration cell with an XM-100 A filter and 200ml 0.3 M NaCl in 0.1 M Naphosphate buffer, pH 6.6, was passed through the cell followed by 350ml 0.15 M NaCl in 2 . 9 m ~ veronal-HC1 buffer, pH 7.35. The concentration of fibrinogen in the two preparations were determined by the method of Blomback et al. (1972) except that fibrinogen was precipitated by adding 2 vol. of 20% (wlv) ice-cold trichloroacetic acid. The concentration of fibrinogen in preparation I was 1.51 mg/ml, in preparation I1 2.85 mg/ml and in preparation I11 2.81mg/ml, or 4.44pM, 8.38pM and 8.26 pM , respectively, assuming a molecular weight of 340 000 (Blomback, 1967). The protein in the different preparations was 97-98% clottable, using bovine thrombin (Jakobsen & Kierulf, 1973). Purification of equine myoglobin

Preliminary experiments showed that commercial myoglobin contained large amounts of free

QUANTITATION OF AMINO-TERMINAL RESIDUES

glycine which was removed as follows: 16mg myoglobin was dissolved in 4ml lOmM Naphosphate buffer, pH 6.5, containing 0.63 M NaC1, and submitted to gel filtration on a Sephadex G-25 column (1.6 x 21 cm). The fractions absorbing at 409nm were pooled, dialyzed against 10 mM Na-phosphate buffer, pH 6.5 and submitted twice to ion-exchange chromatography on a CM-32 Whatman cellulose column (2.2 x 3.5 cm) equilibrated to the same buffer. In the first run myoglobin was eluted with the same buffer. In the second run myoglobin had increased affinity for the resin and was eluted with 0 . 2 ~Na-phosphate buffer, pH 6.5. The myoglobin eluate was passed through a UM-10 Amicon filter with 16Oml 0.2 M Na-phosphate buffer, pH 5.8, containing 1 M NaCl. Then 2OOd 0.05M (NH4)2c03, adjusted to pH 8.6 with 6 M acetic acid was passed through the cell. By the method of Waddell (1956) with bovine serum albumin as standard the protein concentration in the myoglobin solution was found to be 0.674mg/ml, or 39.7pM using the molecular weight of 16951 for myoglobin (Dautrevaux et al., 1969). Calibration o f the labelling assay The assay was calibrated by using the standard solution of unlabelled amino acids. As shown by Brown & Perham (1973) there is a linear relation between the 'H/'T ratio and the amount of unlabelled amino acid added. The calibration curve of each amino acid was obtained as follows: the amount of unlabelled amino acid to be analyzed was mixed with 101.11 of the solution of the 14C-amino acids in the test tube. After drying, 1 0 0 4 coupling buffer (1 5 ml pyridine, 10ml water and 1.2 ml dimethyl-allylamine with pH adjusted to 9.8 with 10% (v/v) trifluoroacetic acid) was added. A similar buffer of pH 9.0 is used in the Edman degradation of proteins (Doolittle, 1965). After the addition of lop1 'H-dansyl chloride, the tube was covered with Parafilm and incubated at 37" in the dark. After 30min the reaction was stopped by adding 1 0 4 99% formic acid, then the mixture was dried and the dansylamino acids in the residue were separated by thin-layer chromatography on polyamide sheets,

usually after extraction of the samples (see quantitation of amino-terminal alanine in fibrinogen). Chromatography on polyamide sheets

The dansyl amino acids were dissolved and applied as described by Brown & Perham (1973) on 5 x 5cm polyamide sheets. The bulk of the sample was usually applied. The chromatogram was developed according to Woods & Wang (1967) with the modification described previously (Flengsrud, 1976). Streaking appeared in some cases after solvent 3, but this was corrected by a rerun of the chromatogram in solvent 1 and sometimes also a rerun in solvent 3 was advisable. A metal framework with spacers between the sheets allowed several chromatograms to be run simultaneously. The separated dansyl amino acids were prepared for counting as described by Brown & Perham (1973) with the exception that hyamine hydroxide in methanol (0.2 ml of 10% (w/v) (Joseph & Halliday, 1975)) was used for elution, and that 5 ml of scintillation liquid (Instagel or Dilusolve) was added. The samples were counted for 'H and 14C in a Packard TriCarb Liquid Scintillation Counter. Background counts were determined by repeating the above procedure using a piece of polyamide sheet cut from a non-fluorescent area of the chromatogram. A pure 'H-sample was prepared by cutting out a piece of Dan-OH spot from a chromatogram. A pure 14C-sample was prepared by mixing 3 p1 of 14C-alanine solution (2.45pCilml in water) with 0.2ml of the solution used for elution and 5 ml Instagel. No quenching of 'Hdansyl chloride by increasing amounts of unlabelled dansyl chloride was observed at levels of the latter below 4 nmol. Calculation of the 'Hf 14Cratio

If the fraction of 3H-counts in the 14C-channel is negligible, the ratio 'H/14C becomes (Perham, 1974): 'H/14C =

T' - B~

-€ C' - Bc

T' and C' are the counts in the 3H- and 14Cchannels, respectively, BT and Bc the corresponding background counts and E is the 50 1

R. FLENGSRUD TABLE 1

Calibration of the labelling assay The constants of the Linear relationship: 3I/'"C = a x + b , are given, where x is the amount (in nmol) of

-

unlabelled amino acid added. The equations were calculated on the basis of 4-10 values in the range 0.011.7 nmol amino acid Hdansyl chloride

Amino acid

I I I1 I1 Ill IV

constant fraction of the ''Ccounts that appears in the 3H-channel. The punched data were fed into a Wang 600 computer (Wang Lab., Tewksbury, Mass., U.S.A.) for calculation of the 3H/'4C-ratio and linear regrtssion analysis was carried out. The equations obtained in the calibration of the assay are given in Table 1. An example of the use of these equations for calculation yield from dansylation of protein is shown. A sample of 2.22 nmol fibrinogen gave the corrected 3H/'4C-ratio 1.24 using 3H-Dan-C1 batch I. When this value is used in the equation for glycine in Table 1, this equals 0.824nmo1, which is 18.5% of the theoretical value of 4.44 nmol Lmino-terminal alanine in 2.22 nmol fibrinogen.

a

b

1.43 1.51 2.1 8 1.97 1.30 2.24

0.021 0.0013 0.076 0.038 -0.151 - 0.405

f

S.D. (%)

3.9 8.0 3 .I 0.9 3.0 7.1

Quantitation o f amino-terminal alanine in fibrinogen

Fibrinogen was precipitated with 2 vol. ice-cold 20% (w/v) trichloroacetic acid (TCA). The mixture was left in an ice-bath for 30min and then centrifuged for 5min at 8000g and 4O. The supernatant was carefully removed and discarded. 1 ml acetone was added to the residue and the mixture was shaken on a Vortex mixer and centrifuged as above. This operation was repeated three times. The residue was dried, oxidized with performic acid and then lop1 of the mixture of '*C-labelled glycine, alanine and tyrosine was added, followed by drying. Afterwards, 1 7 0 ~ 1of the coupling buffer was added and the tube covered with Parafilm and incubated at 50" for 10min TABLE 2

Yield o f dansylation o f free alanine and glycine

The yield of dansyl derivates of glycine and alanine was studied using pH 9.0, and 9.8 in the coupling buffer. For comparison the yield in another procedure (Gros & Labouesse, 1969) was studied. The residues after coupling and drying were applied quantitatively on 20 x 2 0 cm Silicagel G layers (0.3 mm thick) and chromatographed in solvent systems d and b (Deyl & Rosmus, 1965). The chromatograms were scanned in a Perkin-Elmer MPF3A Fluorescence Spectrophotometer equipped with thin-layer chromatography accessories using an excitation wavelength of 329 nm and an emission wavelength of 521 nm with slit widths of 4 n m . The peaks were integrated manually by triangulation. The results are given in Table 2. 502

Yield of dansyhtion of free alanine and glycine Procedure

Fluorescence (arbitrary units) of Dansyl-alanine

Dansyl-glycine ~

This work pH 9.0 pH 9.5 pH 9.8 Gros & Labouesse ( 1 969).

3.6 f 0.9' 7.9 f 1.8 8.6 f 1.2

11.2 f 3.2' 15.4 f 4.0 12.8 f 2.6

8.7

14.1 f 4.2

f

1.8

5nmol each of alanine and glycine were dansylated together and quantified by fluorescence scanning of the thin-layer chromatogram (See Experimental procedures and results). 'Mean of four experiments. All other values mean of five experiments.

QUANTITATION OF AMINO-TERMINAL RESIDUES

to ensure complete solubilization. The tube was cooled to room temperature and 3 0 p l of 3H-dansyl chloride solution was added. The tube was covered with Parafilm, shaken and incubated in a water bath at 37" in the dark. After 30min, lop1 99% formic acid was added to stop the reaction. The mixture was dried and hydrolysed in 0.5ml 6~ HCl under vacuum for 4 h at 110" in an oilbath (Gros & Labouesse, 1969). The hydrolysate was dried and 300pl 0.5% (v/v) NH3 and 500-6OOpl benzene were added to the residue and mixed on a Vortex mixer. The phases were allowed to separate and the upper phase (benzene) was removed and discarded. This extraction was repeated 4-5 times to remove dansyl-amide. The remaining aqueous phase was adjusted to pH 3.5 by adding 25 pl 6 N acetic acid, the pH being monitored using pH-paper. The solution was then extracted 5-6 times with 500-6OOpl diethyl ether (Gros & Labouesse, 1969). The ether phases were combined and dried by the application of hot air to the outside of the tube. Any benzene carried over with the ether was evaporated in a stream of nitrogen. Chromatography on polyamide sheets and calculation of the ratio 3H/'4C were performed as described above. The yield of the coupling with amino-terminal tyrosine in fibrinogen was too low to be quantified. Tyrosine was recovered as a-dansyl tyrosine in very poor yield and in the same form from dansylation of free tyrosine at pH 9.5-9.8. Using 0.1 M NaHC03, pH 9.8 or by the procedure of Gros & Labouesse (1 969), bis-dansyl tyrosine was the main product. This difference and possibly also the low yield of dansyl tyrosine from fibrinogen, may be caused by an effect of pyridine on the dansylation of tyrosine. Low yields of amino-terminal tyrosine were also reported by Milstein (1966). The yield of amino-terminal alanine was studied using pH 9.0, 9.5 and 9.8 in the coupling buffer (Table 3). Using pH 9.8, a linear relation was found between the 3H/14C ratio in dansyl alanine and the amount of fibrinogen up to at least 2.6nmol (Fig. 1A). The average yield in 10 experiments in the range 1-2.6 nmol was 18% (S.D. = f7.4%). When performic acid oxidation of fibrinogen was omitted the yield was 0.8%. The addition of other 14C-amino

acids (valine, leucine and lysine at 2.5-5 nCi) to the coupling mixture did not influence the yield. Several parameters were varied in an attempt to increase the yield. The water/ pyridine ratio seemed to be critical. Too much water gave lower yields, too much pyridine decreased the dansylation of free amino acids. Other buffer substances, like N-ethylmorpholine, gave the same yield. No significant change in the yield was seen when the concentration of 3H-dansyl chloride was doubled in the coupling assay. Quantitation of amino-terminal glycine in m y oglo bin

Since horSe myoglobin contains neither cysteine nor cystine residues (Dautrevaux et al., 1969) the performic acid oxidation step was omitted. The amount of myoglobin to be analyzed was mixed with 10 pl of the mixture of 14C-labelled alanine, glycine and tyrosine. The coupling was carried out as described for calibration of the labelling assay and the residues after drying of the reaction mixture were treated as described for the amino-terminal analysis of fibrinogen. A linear relationship between the amount of myoglobin used and the 3H/'4C ratio of the separated amino-terminal glycine was found both at pH 9.5 and at pH 9.8 (Fig. 1B) in the coupling buffer. The average yield of 11 myoglobin experiments in the range 0.2-4.57 nmol at pH 9.5 was 30% ( S . D . = +3.0%). At pH 9.8 the yield was 31% (S.D. =+3.4%). With a different batch of 14C-glycine the average corrected 3H/14C ratio and standard deviation for six determinations on 25pmol samples of the same myoglobin solution was 0.0516 f 0.0094, for six determinations on 50pmol 0.0584 f 0.0038 and for five determinations on 0.1 nmolO.0646 f 0.0037. Quantitation o f amino-terminal aspartic acid in IgA

The protein was precipitated with 2vol. icecold acetone and the mixture was left at -20" for 1 h (Neuhoff, 1973) and then centrifuged for 1Omin at 8000g at 4". The supernatant was carefully removed and discarded. The precipitated protein was oxidized with performic acid and lop1 of the amino acid mixture (CFB 104) was added. The labelling 503

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Amount o f f i b r i n o g e n (nmoles)

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Relationship between the ratio 'H/"C in dansyl-alanine and the amount of fibrinogen added to the dansyl mixture (A), between the 'H/"C in dansyl-glycine and the amount of myoglobin added (B), and between the ratio H/" C in dansylaspartic acid and the amount of IgA added (C). Each sample was counted 5 times (1Omin) except the myoglobin analyses, which were counted once (10min). The lines are drawn on the basis of linear regression analysis of the data (A: S.D.= f 7.4%. B: S.D. = i 3.4%, C: S.D. = f 10.3%). Experimental details are given in the text. Fibrinogen preparation I was used, 'Hdansyl chloride batch I (A & B) and 'Hdansyl chloride batch IV (C). In A, c.p.m. for 'H was 51-383 and for "C 80.0-591.8. The backgrounds were 15.3 and 12.7 c.p.m., respectively. In B, c.p.m. for 'H was 43.1-873.9 and for 143.9-441.4. The backgrounds were 5.8 and 14.6 c.p.m., respectively. In C, c.p.m. for 'H was 71-751.1,and for 14C 57.9-618.5. The backgrounds were 16.8 and 25.7 c.p.m., respectively.

(0.

FIGURE 1 Quantitation of amino-terminal residues of fibrinogen (A), myoglobin (B) and IgA

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QUANTITATION OF AMINO-TERMINAL RESIDUES

procedure was carried out as described for calibration of the assay with the exception that 201.11 of the 3H-dansyl chloride was added. Further treatment of the labelled protein was done as described for the quantitation of amino-terminal alanine in fibrinogen. The results are given in Fig. 1C. The average yield in the experiments was 49.3% ( S.D. = f 10.3%).

Each sample was then treated as described for quantitation of amino-terminal aspartic acid in IgA with the exception that 501.11 of the 14Camino acid mixture containing glycine, alanine and tyrosine was added. The results are given in Fig. 2.

Quantitation o f free amino acids in the protein preparations

The different techniques used in sequence analysis of peptides and proteins have been reviewed by Deyl (1976) who stated that 41 types of derivatives have been described. Only few of these have been used in quantitative analysis. Most common has been the Edman procedure (Edman, 1960; Eriksson & Sjoquist, 1960). The use of radiochemical derivatives of phenylisothiocyanate has been described (Burrell et al., 1975; Jacobs & Niall, 1975). Other reports on quantitative analysis of p e p tides and proteins have described the use of pivalyl or benzoyl chloride (Cavadore et al., 1974) and cobalt (110 chetates (Bentley, 1976). High performance liquid chromatography of different amino acid derivatives may also be useful in such quantitative analysis (Yosida etal., 1975). This report describes the use of dansyl chloride in the quantitative analysis of proteins and the present results show that the method described gives a linear relationship between the ratio 'H/14C in the separated amino-terminal residues and the amount of protein added (Fig. 1). The lower detection limit seems to be around 0.1 nmol for myoglobin and 0.20.25nmol for fibrinogen and IgA under the conditions used. Since even smaller amounts can easily be seen on the chromatograms the limiting factor in this respect may be the ratio of the specific activities of 3H- and 14C-isotopes. It is not advisable t o lower the concentration of 14C-isotopes further and consequently the sensitivity could be increased only by using in the assay a 3H-labelled dansyl chloride of higher specific activity. The yields of the coupling of the different amino-terminal residues relative to the corresponding free amino acids show considerable variation. Consequently, the method is not suited for quantitation of moles of aminoterminal amino acids per mole of protein.

This was performed as described for the aminoterminal residues of the proteins except that the hydrolysis was omitted. The main contaminating free amino acids were glycine and alanine in the fibrinogen preparations and glycine in the myoglobin preparation. In the different preparations the following amounts of free amino acids were found per mole of protein: Preparation I of fibrinogen contained 0.23 mol glycine and 0.065 mol alanine, preparation I1 of fibrinogen contained 0.1 1 mol glycine and the myoglobin preparation contained 0.058 mol glycine. Determination o f the increase of aminoterminal glycine in fibrinogen during the thrombin action

The method for quantitation of amino-terminal residues in fibrinogen was originally designed for a study on the thrombin-fibrinogen reaction. Since the fibrinopeptides released in the thrombin action are soluble in TCA (Lorand, 1953), precipitation of fibrinogen in TCA should theoretically allow simultaneous measurement of the decrease in amino-terminal alanine and the appearance of glycine. Preliminary kinetic experiments on the thrombinfibrinogen reaction indicated, however, that it was impossible to wash the liberated fibrinopeptides quantitatively out of the precipitated fibrinogen. Consequently, it was decided to measure only the increase in amino-terminal glycine, which was done in the following way: 2.8 ml of the fibrinogen solution (preparation 111) was kept at 37" in a water bath and mixed with 201.11 of the thrombin solution. Immediately 0.2 ml aliquots were taken out to separate glass-tubes and incubated at different times at 37'. The reaction was stopped by putting the tubes on ice and adding 0.8 ml ice-cold acetone.

DISCUSSION

505

R. FLENGSRUD However, the kinetic study on the thrombinfibrinogen reaction does suggest that the method is suitable for studies on specific proteolytic reactions. The quantitation of the increase of amino-terminal glycine in fibrinogen (Fig. 2) shows a pattern of limited proteolytic action of thrombin similar t o that found when applying Edman's phenylthiocarbamyl method (Blomback, 1958; Abildgaard, 1965; Jakobsen & Kierulf, 1973; Blomback er al., 1976). Although this curve has been drawn as a smooth line, it is possible that there should be a deflection in the curve after the coagulation point (Blomback, 1958; Blomback er al., 1976). In this experiment the reaction was not followed to completion but preliminary experiments suggest that a plateau was reached at around 3H/'4C = 0.39. This would imply a yield of amino-terminal glycine in fibrin of 6.3%.

Subtraction of the background value of glycine in fibrinogen would lead to a even lower yield. This very low yield might be explained by a considerable steric hindrance of the coupling reaction and perhaps also by reduced solubility of the performic acid oxidized fibrin in the coupling buffer. This experiment shows that around 50% of the total amino-terminal glycine detected was liberated at the coagulation point. Abildgaard (1965) found that 40% of the total glycine increase had occurred at the coagulation point using human fibrinogen and human thrombin, but this fraction liberated at the coagulation point seems to be very dependent on the reaction conditions (Blomback, 1958). The reproducibility of the method depends on several of the reaction parameters. Some of these have been investigated in the present work and will be discussed. In the experiments

1

I

I

5

10

15

Incubation time (min)

FIGURE 2 Quantitation of the increase of amino-terminal glycine in human fibrinogen during incubation with human thrombin. Relationship between the ratio %/"C in dansyl-glycine in fibrinogen and the incubation time (min) with thrombin at 37". Aliquots of 0.2 ml of the thrombin-fibrinogen mixture (fibrinogen preparation 111) were removed after different times of incubation, the reaction stopped by incubation on ice and addition of 4 vol. icecold acetone and each sample submitted to amino-terminal analysis. Further experimental details are given in the text. Each sample was counted 5 times (10min) and c.p.m. for 'H was 99-582.5 and for 'T129.7882.5. The backgrounds were 17.9 and 18.1 c.p.m., respectively. 'Hdansyl chloride batch 111 was used. The arrow indicates formation of visible precipitate. 506

QUANTITATION OF AMINO-TERMINAL RESIDUES TABLE 3 Yield of amino-terminal alanine in fibrinogen pH in coupling buffer

Ratio 3H/14C

Alanine detected (nmol)

Yield of aminoterminal alanine

(%I 9 .O 9.5 9.8

4.60

2.22 1.68

2.32 1.11 0.83

45.9 22.0 16.6

'H-Dansyl chloride (batch 11) and fibrinogen (preparation 11) (2.51 nmol in each experiment) were used. For procedure see Experimental procedures and results.

with quantitation of amino-terminal alanine it was found that the presence of only small amounts of TCA during the coupling gave false high yields since it lowered the yield of the internal standard 14C-amino acid. At pH 9.0 both glycine and alanine are largely protonated and their dansylation when added as internal standards is less than half of that at pH 9.8 (Table 2). Since the amino-terminal residues in a protein usually have a lower pK,-value the 3H/14C ratio will increase and give the false impression (Table 3) that the yield of aminoterminal residues is higher at pH 9.0 than it is at pH 9.8. It is therefore preferable to carry out the coupling at pH 9.8 and it is important that pH is carefully controlled when comparisons are to be made. Since the internal standard does not correct for losses in the coupling procedure it is very important that the coupling conditions are carefully controlled. It is recommended that the incubation time in the coupling assay is exactly 30min since the incubation time does seem to influence differently the dansylation of free amino acids (Neuhoff, 1973) and amino-terminal residues in proteins (Gros & Labouesse, 1969). Pyridine is reported to reduce the yield of dansylation reactions (Gray, 1972). In my hands dansylation of free amino acids by the present method is essentially similar to that obtained by the method of Gros & Labouesse (1969) (Table 2). Since the present method utilized 14C-amino acids as internal standard, the method of Gros & Labouesse (1969), which involves precipitation after dansylation, could not be used. After the hydrolysis step the internal standards will correct accurately for losses in the

procedure. However, another critical point in the procedure is the chromatographic separation. It is always advisable to extract dansylamide with NHJ/benzene since a high amount of dansyl-amide may contaminate the neighbouring spots in the chromatograms. This is probably the cause of the relatively high s .D .-value in the standard curve for alanine obtained with dansyl chloride batch I, where this extraction was omitted. In spite of the internal standard, the amount of sample applied to each polyamide sheet is not completely without importance. Too small an amount will give c.p.m.-values that are too low and too large an amount may give incomplete separation of the dansyl spots, although with the concentrations used in this work the bulk of the sample was usually applied. As discussed by Brown & Perham (1973) it is believed that the main contribution to the differences in s .D.-values comes from the chromatographic separation. The difference in quality of the polyamide sheets from different origins discussed previously (Joseph & Halliday, 1975) was also observed by this author. It is also important to determine the amount of free amino acids in the protein preparations when very small amounts of protein are used and especially in the analysis of glycine. Although this method has been demonstrated on three different amino-terminal residues only, it is probably that it is applicable to most other residues as well. However, the procedure involves ether extraction from a water phase which will leave the following dansyl derivates in the water phase: Dan-Arg, a-Dan-His, e-Dan-Lys, o-Dan-Tyr, Dan-cysteic acid, mono-Dan-cystine and Dan-OH (Gros & 507

R. FLENGSRUD

Labouesse, 1969). Chromatogaphic separation of the derivates in the water phase is difficult because of the large excess of dan-Oh. Therefore the present method is not applicable to arginine, histidine and cysteine as aminoterminal residues unless a method for the separation of their densyl-derivates from dan-Oh in the water phase is found. In addition, the yield of amino-terminal tyrosine in fibrinogen was too low to be quantified, but it is possible that this problem could be solved by increasing the concentration of radioisotopes in the coupling assay. The hydrolysis conditions used will lead to the destruction of tryptophan (Cros & Labouesse, 1969) but the use of other acids for the hydrolysis will allow the detection of amino-terminal tryptophan (Flengsrud’, 1976; Giglio, 1977). It should be kept in mind that in the case of amino-terminal residues valine, leucine and isoleucine the hydrolysis time should be increased to 18h (Cros & Labouesse, 1969). In conclusion, the present method has the advantage of good sensitivity and reproducibility in the quantitation of certain aminoterminal residues. ACKNOWLEDGEMENT The technical assistance of Renie Johansen is gratefully acknowledged, as are the valuable discussions with Clive Little, Hans Prydz and Reidar Wallin duTing this work and the financial support of the Norwegian Council on Cardiovascular Diseases.

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