Journal of Chromatography, 530 (I 990) 29-31 Biomedical Applications Elsevier Science Publishers B.V., Amsterdam CHROMBIO. 5344 High-performance liquid chromatography of rat and mouse islet polypeptides: potential risk of oxidation of methionine residues during sample preparation’ S. LINDE*, J. H. NIELSEN, B. HANSEN and B. S. WELINDER Hugedorn Research Laborutorq; Niels Steensensvt~j 6. DK-2820 Gentqfte (Denmark) (First received November 3rd, 1989; revised manuscript received March 26th, 1990) ABSTRACT After preparative high-performance liquid chromatography of mouse islet culture medium, concentrat- cd on disposable C,, cartridges (Sep-Pak), an unexpected insulin immunoreactive peak cluting earlier than mouse insulin I and II was detected. Molecular mass determination by mass spectrometry supported its suspected identity as methionine sulphoxide insulin II. We have examined the formation of Met-O deriv- atives of insulin II, glucagon and pancreatic polypeptide during sample preparation (Sep-Pak and Speed- Vat concentrating). The oxidation of methionine residues was found to depend very much on the buffer, the organic modifier and the procedure. In particular the use of methanol-trifluoroacctic acid resulted in extensive oxidation. The oxidation could be minimized by adding 2 mM dithiothreitol to the buffer and by degassing and/or nitrogen-bubbling of the buffer. Minimal formation of Met-O derivatives is important for the quantitation of mcthioninc-containing polypeptides. INTRODUCTION Studies of the biosynthesis of proinsulins and their conversion into insulins and C-peptides in the rat and mouse endocrine pancreas depend on accessible reversed-phase high-performance liquid chromatographic (RP-HPLC) analyses capable of separating all the polypeptides involved. We have recently described two HPLC systems that can separate the two non-allelic insulins (1 and II) from rat and mouse, as well as the two C-peptides (I and II), and the two proinsulins (I and II) [l]. I n order to identify the individual mouse polypeptides by amino acid analysis and micro-sequencing [l] we concen- trated medium from cultured mouse islets on disposable Cl* cartridges (Sep- Pak). In some cases this procedure resulted in an unexpected peak which reacted as insulin by radioimmunassay after RP-HPLC separation of the concentrated medium. Since insulin II, in contrast to insulin I, contains a methionine residue
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`in
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`a Presented in part at the 9th International Symposium on HPLC of Proteins, Peptides and Polynucleo- tides, Philadelphia, PA, November 6-8, 1989. The majority of the papers presented at this symposium have been published in J. Chromatogr., Vol. 512 (1990). 037%4347/90/%03.50 0 1990 Elsevier Science Publishers B.V.
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`CFAD Exhibit 1052
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`30
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`S. LINDE el al.
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`position 29 of the B-chain, oxidation to methionine sulphoxide in insulin 11 might
`have happened during sample preparation [2—5].
`The aim of the present study was to investigate the influence of buffer compo-
`nents, organic modifiers and isolation procedures on the formation of Met—O
`derivatives of insulin 11, as well as of glucagon and pancreatic polypeptide, in
`order to minimize the oxidation of the methionine residues.
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`EXPERIMENTAL
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`30 S. LINDE et al. position 29 of the B-chain, oxidation to methionine sulphoxide in insulin II might have happened during sample preparation [2-51. The aim of the present study was to investigate the influence of buffer compo- nents, organic modifiers and isolation procedures on the formation of Met-O derivatives of insulin II, as well as of glucagon and pancreatic polypeptide, in order to minimize the oxidation of the methionine residues. EXPERIMENTAL Reagents Trifluoroacetic acid (TFA, Peptide Synthesis grade) was from Applied Bio- systems and acetonitrile (HPLC grade S) was from Rathburn. All other chemicals were of analytical-reagent grade. Distilled water was purified on a Millipore Mil- li-Q plant, and all buffers were filtered (0.45 pm, Millipore) and degassed by vacuum/ultrasound before use. Standards As a source of rat insulin I and II and C-peptide 1 and II, medium from newborn rat islet cells cultured in RPM1 1640 supplemented with 2% human serum in the presence of I ,ug/ml human growth hormone was used. The medium contained 44 pg/ml of insulin 1 and II and equimolar amounts of C-peptide I and II. Rat pancreatic polypeptide was obtained from Penninsula and porcine gluca- gon from Sigma. Rat insulin II was prepared from the culture medium using preparative HPLC. Chromatography The HPLC system consisted of Waters M6000 A pumps, a WISP 7 lOA, a 660 solvent programmer, a 730 data module and a Pye Unicam LC-UV detector. The column was LiChrosorb RP-18, 5 pm particle size, 2.50 x 4.0 mm I.D. (Merck), eluted at 1.0 ml/min with a linear gradient of acetonitrile (30 to 36%) in 0.1% TFA during 60 min. The column eluate was monitored at 210 nm, peaks were collected either manually or by collecting 0.5-min fractions in a FRAC 300 frac- tion collector (Pharmacia). All separations were carried out at room temperature. Sample preparation using Sep-Pak Disposable Cl8 cartridges (Sep-Pak, Waters) were used. The organic eluent (B) contained 90% (v/v) of the organic modifier, which was acetonitrile, 2-propa- no1 or methanol. The aqueous eluent (A) was water, 1 A4 acetic acid or 0.1% TFA. The Sep-Pak cartridge was flushed with IO ml of B and 10 ml of A, and the sample was applied, followed by washing with 10 ml of A and elution with 1.5 ml of B into an Eppendorf tube. The sample was concentrated in a Speed-Vat con- centrator (Savant).
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`Chromatography
`The HPLC system consisted of Waters M6000 A pumps, 21 WISP 710A, a 660
`solvent programmer, a 730 data module and a Pye Unicam LC-UV detector. The
`column was LiChrosorb RP—l8, 5 am particle size, 250 X 4.0 mm l.D. (Merck),
`eluted at 1.0 ml/min with a linear gradient of acetonitrile (30 to 36%) in 0.1%
`TFA during 60 min. The column eluate was monitored at 210 nm, peaks were
`collected either manually or by collecting 0.5—min fractions in a FRAC 300 frac-
`tion collector (Pharmacia). All separations were carried out at room temperature.
`
`Reagents
`Trifluoroacetic acid (TFA, Peptide Synthesis grade) was from Applied Bio-
`systems and acetonitrile (HPLC grade S) was from Rathburn. All other chemicals
`were of analytical-reagent grade. Distilled water was purified on a Millipore Mil-
`li-Q plant, and all buffers were filtered (0.45 pm, Millipore) and degassed by
`vacuum/ultrasound before use.
`
`Standards
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`As a source of rat insulin I and II and C-peptide I and II, medium from
`newborn rat islet cells cultured in RPMI 1640 supplemented with 2% human
`serum in the presence of 1 ng/ml human growth hormone was used. The medium
`contained 44 pg/ml of insulin 1 and II and equimolar amounts of C—peptide I and
`11. Rat pancreatic polypeptide was obtained from Penninsula and porcine gluca—
`gon from Sigma. Rat insulin 11 was prepared from the culture medium using
`preparative HPLC.
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`Sample preparation using Sep—Pak
`Disposable C13 cartridges (Sep—Pak, Waters) were used. The organic eluent
`(B) contained 90% (V/V) of the organic modifier, which was acetonitrile, 2—propa-
`nol or methanol. The aqueous eluent (A) was water,
`1 M acetic acid or 0.1%
`TFA. The Sep—Pak cartridge was flushed with 10 ml of B and 10 ml of A, and the
`sample was applied, followed by washing with 10 ml of A and elution with 1.5 ml
`of B into an Eppendorf tube. The sample was concentrated in a Speed—Vac con-
`centrator (Savant).
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`HPLC OF RAT AND MOUSE ISLET POLYPEPTIDES
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`31
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`RESULTS AND DISCUSSION
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`Rudioimmunoassay (RIA) Collected fractions containing TFA-acetonitrile were dried in a Speed-Vat concentrator, and radioimmunological determination of insulin was carried out using rat insulin (Novo) as a standard and anti-mouse insulin antibodies (devel- oped in this laboratory) as previously described [6]. Hydrogen peroxide oxidation A l-ml volume of a solution containing 1% hydrogen peroxide (v/v) in 3 A4 acetic acid was evaporated in the Speed-Vat concentrator in a separate tube, together with 240 pg samples of glucagon, pancreatic polypeptide or insulin 11 dissolved in 3 M acetic acid, as well as rat culture medium diluted 1: 1 (v/v) with 3 M acetic acid. Determination of molecular mass The molecular masses (44,) of the polypeptides and their oxidized forms isolat- ed after HPLC were determined by mass spectrometry (MS). The mass spectra were obtained on a Bio-Ion 20 plasma desorption mass spectrometer [7]. Prior to analysis the samples were applied to nitrocellulose targets prepared on Mylar foil. Spectra were accumulated for 5.106 fission events.
`Isolation and identification of polypeptides and proteins present in very small amounts in complex solutions, such as cell culture media, normally require a concentration step before the HPLC analysis, and especially for large volumes it is convenient to use disposable Sep-Pak C I~ cartridges for sample preparation. Mouse islet culture media (100 ml) were loaded on a Sep-Pak, the cartridge was washed with 10 ml of 30% methanol (thereby eluting several non-peptide culture medium constituents) and thereafter the polypeptides were eluted with 90% methanol. Fig. 1 shows the HPLC analysis of the concentrated medium. Although several sample components with no relevance to the C-peptides and insulins were removed before HPLC, the presence of numerous UV-absorbing components made a direct localization of the islet polypeptides difficult: mouse C-peptide I and II, expected to elute at 25530 min, could not be distinguished from neighbouring components, and only after insulin RIA were the insulins localized in three peaks. Peaks 2 and 3 eluted at the same retention times as rat insulin I and II, respectively (rat and mouse insulins are identical). In order to verify the hypothesis that insulin peak 1 could be methionine sulphoxide insulin II, rat islet culture medium was oxidized with hydrogen perox- ide reported to oxidize methionine to methionine sulphoxide [8] as described above. HPLC analysis of this sample showed that an additional component with a lower retention time (marked with an arrow, Fig. 2) was formed during ox- idation simultaneously with a decrease of the amount of insulin II. The lower
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`S. LINDE er a1.
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`Elution time (min)
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`600
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`Fig. 1. HPLC separation of 40 ml of Sep-Palcconcentrated mouse islet culture medium using a LiChrosorb
`RP—18 column (250 x 4.0 mm I.D.) eluted at a flow—rate of 1.0 ml/min with a Concave acetoniirile gradient
`(gradient 7, 30 to 36%) in 0.1% TFA during 60 min; 0.5—min fractions were collected and dried in a
`Speed-Vac, and the insulin content was determined using RIA (histogram).
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`Ra! islet culture medium
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`Ema
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`0.08
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`E210
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`32 S. LINDE et al. Elution time (min) Fig. 1. HPLC separation of 40 ml of Sep-Pak-concentrated mouse islet culture medium using a LiChrosorb RP-18 column (250 x 4.0 mm I.D.) eluted at a flow-rate of I .O mllmin with a concave acetonitrile gradient (gradient 7, 30 to 34%) in 0.1% TFA during 60 min; 0.5.min fractions were collected and dried in a Speed-Vat, and the insulin content was determined using RIA (histogram). 0.08- 0 W" o- Rat islet culture medium O.O& 0 W" 0-I T Oxidized culture medium + ’ I 1 6 2’0 4,o ZO Elution time (min) Fig. 2. HPLC separation of 50 ~1 of rat islet cell culture medium and 75 ~1 of H,O,-oxidized medium using a LiChrosorb RP-18 column (250 x 4.0 mm I.D.) eluted at a flow-rate of 1.0 ml/min with a linear aceto- nitrile gradient (3&36%) in 0.1% TFA during 60 min.
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`C~c-eptideII
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`InsulinI
`insulinIi
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`C-peptideI
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`Oxidized culture medium
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`4-
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`Elution time (min)
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`Fig. 2. HPLC separation of 50 /11 of rat islet cell culture medium and 75 ul of HlO2—oxidized medium using
`a LiChrosorb RP-18 column (250 x 4.0 mm I.D.) eluted at a flow—rate of 1.0 ml/min with a linear aceto-
`nitrile gradient (3(L36%) in 0.1% TFA during 60 min.
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`HPLC OF RAT AND MOUSE ISLET POLYPEPTIDES 33 retention time was expected since the Met-O derivatives are reported to have higher polarity [lo]. The retention time of this newly formed component relative to that of insulin II could not directly be compared with that of peak 1 in Fig. 1, since different acetonitrile gradients were used (see legends to Figs. 1 and 2). The identification of rat insulin I and II (Fig. 2, upper panel) was based on amino acid analysis and sequencing, as well as by RIA as previously described [I]. Likewise, oxidation of other methionine-containing islet polypeptides resulted in additional peaks with lower retention times as shown for glucagon (Fig. 3, two additional peaks) and pancreatic polypeptide (Fig. 4). The additional peaks (marked with arrows in Figs. 2-4) as well as the authentic polypeptides were collected after HPLC and subjected to MS. If necessary, the samples were con- centrated in the Speed-Vat concentrator before MS. The resulting molecular masses are shown in Table I, together with the theoretically calculated values. In the case of glucagon, two additional peaks were detected (Fig. 3) that had the same molecular mass (Table I), although only one methionine residue is present. Two different methionine sulphoxide forms exist. the D- and L-forms. but it is 0.08 Glucagon Elution time (min) Fig. 3. HPLC separation of glucagon (2 pg) and H,O,-oxidized glucagon (3 /Lg). Conditions as in Fig. 2.
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`34 S. LINDE et nl.
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`1
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`Rat pancreatic polypeptide (PP) Oxidized rat PP
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`Elution time (min) Fig. 4. HPLC separation of rat pancreatic polypeptide (1 pg) and H,O,-oxidized pancreatic polypeptide (2 pg). Conditions as in Fig. 2. TABLE 1 RELATIVE MOLECULAR MASSES OF POLYPEPTIDES AND OXIDIZED POLYPEPTIDES DE- TERMINED BY MS M, found M, calculated” Insulin II 5801 .h 5796 Oxidized insulin II 5818.5 5812 Glucagon 3485.7 3483 Oxidized glucagon (1) 3503.1 3499 Oxidized glucagon (2) 3501.9 3499 Pancreatic polypeptide (PP) 4396.4 4399 Oxidized PP 4416.9 441.5 “The calculated molecular masses of the Met-O derivatives were derived by adding 16 to the known molecular masses of the individual polypeptides.
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`HPLC OF RAT AND MOUSE ISLET POLYPEPTIDES 35 TABLE II AMOUNTS OF OXIDIZED GLUCAGON (G), PANCREATIC POLYPEPTIDE (PP) AND INSULIN II (I,) DERIVATIVES FORMED DURING SAMPLE PREPARATION Solvents: AcOH = acetic acid; MeOH = methanol: isoPrOH = 2-propanol: MeCN = acetonitrile. Sample preparation Solvent Oxidized G Oxidized PP Oxidized I, (X) (%) (%) Speed-Vat Water Speed-Vat 3 M AcOH Speed-Vat 0.1% TFA Speed-Vat 90% MeOH-l M AcOH Speed-Vat 90% MeOH-0.1% TFA Speed-Vat 90% MeCN-1 M AcOH Speed-Vat YO% MeCN-0. I % TFA Speed-Vat 90% isoPrOH-1 M AcOH Speed-Vat 90% isoPrOH-0. I % TFA Sep-Pakh 90% MeOH-H,O Sep-Pak 90% MeOH- M AcOH Sep-Pak 90% MeOH-O.l% TFA Sep-Pak 90% MeCN-H,O Sep-Pak 90% MeCN-I A4 AcOH Scp-Pak 90% MeCN-O. 1% TFA Sep-Pak 90% isoPrOH-H,O Sep-Pak 90% isoPrOH-I M AcOH Sep-Pak 90% isoPr0H-O. 1% TFA
`6 33 2 2 1 1 6 5 63 0 9 3 17 24 42 _
`I 42 51 3 3 4 2 6 14 8 12 16 44 17 46 89 79 0 31 14 51 25 47 16 29 18 40 27 63 “Calculated from UV areas (210 nm) after HPLC. bAs described in Experimental. unlikely that these two forms could be separated in the present HPLC system. Since the M, values of these peaks were higher (ca. 16) than that of the parent compounds and since the formation of Met-O derivatives of rat and mouse in- sulin II and proinsulin II during storage and sample treatment [24], as well as of other methionine-containing polypeptides [ 1 l-141, has been described to occur under similar conditions to those reported here, they most probably represent Met-O derivatives of insulin 11, glucagon and PP. In the case of glucagon and corticotropin-releasing factor, the formation of Met-O derivatives results in re- duced biological activity [ 11,121. The formation of Met-O derivatives was found to depend very much on the actual procedure and buffers, as well as the Sep-Pak cartridge used (Table II). In particular the use of 0.1% TFA-methanol resulted in extensive modification. Furthermore, the amount of modified polypeptide was inversely related to the amount of polypeptide used (exemplified by glucagon): Sep-Pak purification us- ing TFA-acetonitrile of 4, 10 and 40 pg resulted in 23, 7 and 3% of oxidized glucagon derivatives, respectively. In accordance with published results [4,15] for pre-purification of transfected AtT20 cells. we observed that the use of TFA-
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`36 S. LINDE et al. TABLE Ill AMOUNTS OF OXIDIZED GLUCAGON (G), PANCREATIC POLYPEPTIDE (PP) AND INSULIN IT (TJ DERIVATIVES FORMED DURING SAMPLE PREPARATION WITH ADDITION OF 2 mM ASCORBIC ACID (AA) OR 2 mM DITHIOTREITOL (DTT) Sample preparation Solvent Additive Oxidized (%) Oxidized PP (%) Oxidized I, (%) Speed-Vat Speed-Vat Speed-Vat Speed-Vat Speed-Vat Speed-Vat Sep-Pakb Sep-Pak Sep-Pak 3 M AcOH 3 M AcOH 3 M AcOH 90% MeOH-O.l% TFA 90% MeOH-O.l% TFA 90% MeOH-0. I % TFA 90% MeOHa. 1% TFA 90% MeOHa. 1% TFA 90% MeOH@l% TFA _ AA DTT _ AA DTT _ AA DTT
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`2 _ _ _ - _ 1 33 42 51 40 47 0 3 28 63 61 79 41 21 _ 7 13 x “Calculated from UV areas (210 nm) after HPLC. *As described in Experimental. acetonitrile-methylene chloride, resulted in extensive formation of Met-O deriv- atives (data not shown). In order to minimize the oxidation, we incorporated 2 mM ascorbic acid (AA) or 2 mA4 dithiothreitol (DTT) in the buffers during sample preparation (Table III). Only DTT effectively protects the methionine group, in accordance with published results [ 161. The Met-O formation could also be decreased by degassing the buffers. This effect was further enhanced by additional bubbling with nitrogen (data not shown). In conclusion, we have shown that Speed-Vat drying and Sep-Pak sample preparation may result in formation of methionine sulphoxide derivatives. The amount of transformation product depends very much on the actual Sep-Pak used and the time for the sample preparation. Thus the amounts reported here can only serve as an indication. In order to minimize the oxidative transformation of methionine we recommend the addition of 2 mM DTT to prevent the HPLC chromatograms being misleading, and to avoid the reduction of biological activ- ity. ACKNOWLEDGEMENTS We thank Dr. Stephen Bayne (Novo-Nordisk A/S) for performing the molec- ular mass determinations, and Ragna Jsrgensen for excellent technical assistance.
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`HPLC OF RAT AND MOUSE ISLET POLYPEPTIDES REFERENCES 37 1 S. Linde, J. H. Nielsen, B. Hansen and B. S. Welinder, J. Chromutogr., 462 (1989) 243. 2 M. LZ Gasparo and M. Faupel, J. Chromatogr., 357 (1986) 139. 3 R. J. Carroll, R. E. Hammer, S. J. Chart, H. H. Swift. A. R. Rubenstein and D. F. Steiner, Proc. Nutf. Acnd Sci. U.S.A., 85 (1988) 8943. 4 D. Gross, A. Skvorak, G. Hendrick, G. Weir, L. Villa-Komaroff and P. Halban, FEBS Lerf., 241 (1988) 205. 5 G. Gold, M. D. Walker, D. L. Edwards and G. M. Grodsky, Diabetes, 37 (198X) 1509. 6 L. G. Heding, Diabetologia, 8 (1972) 260. 7 G. P. Jonsson, A. B. Hedin, P. L. Hakansson, B. U. R. Sundqvist, B. G. S. Save, P. F. Nielsen, P. Roepstorff, K.-E. Johansson, 1. Kamensky and M. S. L. Lindberg. .4na/. CAenr., 58 (1986) 1084. 8 B. Iselin, Helv. Chim. Acta, 44 (1961) 61. 9 N. P. Neumann, Methods Enzymol., II (1967) 485. 10 IJ. Gether, H. Vendelbo Nielsen and T. W. Schwartz, J. Chronrntogr.. 447 (1988) 341. I I W. Vale, J. Spiess, C. Rivier and J. Rivicr, Science, 213 (198 I) 1394. 12 0. Sonne, U. D. Larsen and J. Markussen, Hoppe-Se.vler’s Z. Ph_ysiol. Chem., 363 (1982) 95. 13 R. M. Riggin, G. K. Dorulla and D. J. Miner, Anul. Biochem., 167 (1987) 199. 14 N. A. Farid, L. M. Atkins, G. W. Becker. A. Dinner, R. E. Heiney, D. J. Miner and R. M. Riggin. J. Pharm. Biomrd. Anal., 7 (1989) 185. 15 R. M. Cohen, B. D. Given, J. Licino-Paixao. S. A. Provow, P. A. Rue, B. H. Frank, M. A. Root, K. S. Polonsky. H. S. Tagcr and A. H. Rubenstein, ~Mernholisn?, 35 (1986) 1137. 16 H. V. Huang, M. W. Bond, M. W. Hunkapiller and L. E. Hood, Methods Enz~mol. 91 (1983) 318.