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Vitamin K Dependent Modifications of Glutamic Acid Residues In Proc. Nat. Acad. Sci. USA Vol. 71, No. 7, pp. 2730-2733, July 1974 Vitamin K Dependent Modifications of Glutamic Acid Residues in Prothrombin (proton magnetic resonance spectroscopy/mass spectrometry) JOHAN STENFLO*, PER FERNLUND*, WILLIAM EGANt, AND PETER ROEPSTORFFt * Department of Clinical Chemistry, University of Lund, Malm6 General Hospital, 8-214 01 Malm6, Sweden; t Department of Physical Chemistry II, Lund Institute of Technology, Chemical Center, S-220 07 Lund, Sweden; and t The Danish Institute of Protein Chemistry, DK-2970 Horsholm, Denmark Communicated by Sune Bergstrom, May 9, 1974 ABSTRACT A tetrapeptide, residues 6 to 9 in normal hydrogen on the -y carbon atom by a carboxyl group. This prothrombin, was isolated from the NH2-terminal, Ca2+- work will be described in greater detail elsewhere. binding part of normal prothrombin. The electrophoretic mobility of the peptide was too high to be explained en- tirely by its amino-acid composition. According to 1H MATERIALS AND METHODS nuclear magnetic resonance spectroscopy and mass Isolation of Tetrapeptide. The heptapeptide from normal spectrometry, the peptide contained two residues of modi- fied glutamic acid, -y-carboxyglutamic acid (3-amino-1,1,3- prothrombin (residues 4 to 10) (ref. 13) was first thoroughly propanetricarboxylic acid), a hitherto unidentified amino digested with aminopeptidase M (Sigma) and afterwards with acid. This amino acid gives normal prothrombin the Ca2 +_ carboxypeptidase B (Sigma). A tetrapeptide was isolated binding ability that is necessary for its activation. Obser- from the digest by gel chromatography on Sephadex G-25 vations indicate that abnormal prothrombin, induced by the vitamin K antagonist, dicoumarol, lacks these modi- superfine and obtained in pure form as judged by high voltage fied glutamic acid residues and that this is the reason why electrophoresis at pH 6.5 (electrophoretic mobility relative to abnormal prothrombin does not bind Ca2+ and is non- that of aspartic acid 1.09) and amino-acid analysis (14), which functioning in blood coagulation. gave Glu 2.03, Val 0.97, and Leu 1.01. Prothrombin is a plasma glycoprotein that is activated during Synthesis of Leu-Glu-Glu-Val. Solid-phase technique (15) the process of blood coagulation to the proteolytic enzyme according to the procedures of Stewart and Young was used thrombin. The biosynthesis of prothrombin is vitamin K (16). The peptide was cleaved from the resin by treatment dependent, and deficiency of this vitamin or administration with 45% HBr in acetic acid and purified by gel chro- of the vitamin K antagonist, dicoumarol, gives rise to an matography on a Biogel P-2 column. The peptide was ho- abnormal prothrombin which does not function in blood mogeneous on paper electrophoresis at pH 6.5 (electrophoretic coagulation (1-6). The activation of prothrombin in vivo mobility relative to that of aspartic acid 0.84) and amino-acid requires the binding of Ca2+ (7); abnormal prothrombin does analysis gave Glu 2.07, Val 1.01, and Leu 0.92. not bind Ca2+ (2, 8, 9). During the activation of normal pro- thrombin an NH2-terminal fragment (molecular weight 1H Nuclear Magnetic Resonance Spectroscopy. The measure- approximately 25,000) is split off; the difference between ments were made with a Varian XL-100 spectrometer operat- abnormal and normal prothrombin has been localized to this ing in the Fourier transform mode; the deuterium resonance part of the molecule. Evidence has been produced that the from the solvent D20 (Ciba 99.8 atom percent) served as the difference is due to the lack of certain prosthetic groups in internal lock signal. The chemical shifts are given as 8 values abnormal prothrombin (10-13). and are relative to internal DSS (sodium 2,2-dimethylsila- In an endeavour to define the difference between normal pentane-5-sulfonate); a 60-jssec pulse width and, alternatively, and abnormal prothrombin, the NH2-terminal fragments from 1- and 2-sec acquisition times were used (changing the acquisi- both proteins were isolated and degraded further. A hepta- tion time had no observable effect on the spectrum). Before peptide from normal prothrombin (residues 4 to 10) and a spectra were recorded the lyophilized samples were disssolved corresponding heptapeptide from abnormal prothrombin were in D20 and taken to dryness two or three times in order to isolated by BrCN degradation and trypsin digestion. The substitute all exchangeable hydrogen with deuterium. heptapeptide from normal prothrombin differed from the peptide in abnormal prothrombin in that it had Mass Spectrometry. N-acetylation was done in methanol- corresponding The was then a higher anodal electrophoretic mobility at pH 6.5 (13). acetic anhydride (3:1, v/v). sample subjected to the permethylation procedure of Vilkas and Lederer (17) By extensive proteolytic digestion, the heptapeptide from Morris normal prothrombin was degraded to a tetrapeptide. This but with reduced reaction times as described by (18). The spectra were recorded with a Perkin Elmer 270 mass tetrapeptide, containing residues 6 to 9, still had an abnor- 1000. The mally high anodal electrophoretic mobility at pH 6.5. This spectrometer with a resolution of approximately acid ionizing energy was 70 eV and the ion source temperature was paper reports evidence that each of the two glutamic into the ion source' residues of this peptide are modified by replacement of one 1500. The sample was introduced directly and spectra were recorded repeatedly during slow heating of Abbreviations: DSS, sodium 2,2-dimethylsilapentane-5-sulfonate; the solids inlet probe. Evaporation of the sample occurred in NMR, nuclear magnetic resonance. the temperature range of 150 230°. 2730 Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Modified Glutamic Acid Residues in Prothrombin 2731 RESULTS A tetrapeptide was isolated from a heptapeptide which had the known sequence, Gly-Phe-Leu-Glx-Glx-Val-Arg (13) constituting residues 4 to 10 of normal prothrombin. It was inferred from its amino-acid composition that the tetrapeptide was Leu-Glx-Glx-Val. The yield from the heptapeptide was 64% or approximately 12% from intact normal prothrombin. The tetrapeptide had a high anodal electrophoretic mobility which seemed to be inconsistent with its amino-acid composi- tion even when the Glx residues were assumed to be glutamic acid (13). This inconsistency was confirmed when the mobility of the native tetrapeptide (1.09) was compared with that of a synthetic tetrapeptide Leu-Glu-Glu-Val (0.84). The native thus one extra .1 tetrapeptide possessed at least negative charge, possibly due to a prosthetic group. The 1H nuclear magnetic resonance spectrum of the syn- thetic tetrapeptide (Fig. 1) divides into three readily assign- able areas: the twelve methyl group protons (5 0.8-1.1); the four a protons (6 3.9-4.7); and the remaining 12 methylene and methine protons (b 1.5-2.6). The signals of this latter area appeared in three distinct groups, A, B, and C of Fig. 1, containing approximately (±0.5) 3.3, 4.6, and 4.2 protons, respectively. A tentative assignment of these resonances may _~~~~C A be made with the aid of 1H NMR charts given by Roberts and Jardetzky (19) together with the observation that, in the absence of a secondary or tertiary structure, the 1H NMR | I , I , spectrum of an oligo- or polypeptide is the superposition of 5.0 4.0 3.0 2.0 1.0 0 the spectra of the individual amino acids (19). Thus, the A j3 B FIG. 1. 1H nuclear magnetic resonance spectra (100 MHz) of peak includes the leucine and y protons, the peak the four the synthetic tetrapeptide (below) and native tetrapeptide glutamic acid # protons, and the C peak the four glutamic acid (above); the scale is in ppm (5), and relative to internal DSS. y protons; the valine ,3 protons are spread under both the B Concentrations of 14 mg and 0.9 mg in 0.25 ml of D20 were used and C regions. for the spectra of the synthetic and native tetrapeptide respec- Comparison of the NMR spectrum of the native peptide tively. (Fig. 1) with that of the synthetic tetrapeptide revealed no new resonances. Indeed, with one striking exception, the two substituted, the electron withdrawing power must be suffi- spectra were remarkably similar; while the region of the cient to render the remaining proton exchangeable). These native peptide corresponding to region A of the synthetic substituents, which could contain no inexchangeable hydro- peptide still contained approximately 3.3 protons, the region gens, must be labile enpugh to be removed under the condi- corresponding to B and C indicated loss of approximately four tions used for hydrolysis of the peptide (6 M HCl at 1100 for protons. Since acid hydrolysis of the native tetrapeptide gave 20 hr in vacuo). two glutamic acid residues, the difference could be explained The mass spectrum of the native tetrapeptide (Fig. 2) by the replacement of one or both of the hydrogens on the 'y shows that the conditions used for the derivation did not carbon of the glutamic acid residues (if only one hydrogen is cause N-methylation; the carboxyl groups, however, were AcLeu 4 Me2(COOH)Glu Me2(COOH)GIu - --- Val - fOMel 1 74 100 - 357 ._ 86 I9 0) 128 130 116 299 x5 14 6 l1 42 625 657 0 100 200 300 400 600 m e FIG. 2. Mass spectrum of acetylated and methyl esterified native tetrapeptide obtained at 190°-210°. Downloaded by guest on September 27, 2021 2732 Biochemistry: Stenflo et al. Proc. Nat. Acad. Sci. USA 71 (1974) C ,CH3 HOOC HCOOH HOOC COOH CH CHC s Ct-2 CH2 CH2 A CO-NH-CH-CO- NH-CH- CO-NH- CH-COOH B tK2NN-CH- S I.
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