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[8] Isolation and assay of dolichol and dolichyl phosphate
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[8] I s o l a t i o n a n d A s s a y o f Dolichol a n d D o l i c h y l P h o s p h a t e
By W. LEE ADAm and R. KENNEDY KELLER The isolation and characterization of long-chain prenols t and their monophosphate derivatives are complicated by several factors. First, they are present in small quantities in most tissues examined (5-100/zg/g wet weight). As a result, precise analysis requires the inclusion of a radioactive tracer to monitor purification and correct for losses. Second, the prenols occur naturally in a variety of derivatized states, including acyl and phosphomonoesters of the free alcohols as well as the many glycosylated forms involved in heteroglycan biosynthesis. Since simultaneous analysis of all these derivatives would be a complex task, it is convenient to convert them to a few easily assayable species, namely the free alcohol and the monophosphate ester. For animal tissues this is generally carried out by direct saponification, 2 which completely dissolves the tissue and converts all the dolichol derivatives to dolichol and dolichyl phosphate. With plant tissues (which often contain large amounts of cellulose), it is preferable to extract the prenols with organic solvents prior to saponification. Once extracted, the purification and assay of the prenols and prenyl phosphates have proved a challenge for chromatographers and chemists alike. The chemistry of the long chain prenols is typical for that of allylic and saturated primary alcohols with regards to the hydroxyl end of the molecule. That is, the prenols may be reacted with acyl anhydrides and chlorides to form esters. 3 In addition, they can undergo oxidation to form prenals with a variety of oxidants, such as chromium trioxide-pyridine,4 pyridinium chlorochromate, 5 and manganese dioxide.6 It should be noted that treatment with manganese dioxide forms the basis of a convenient method to distinguish between polyprenol and dolichol, since only the former compound is susceptible to oxidation. An alternative procedure is 1 Nomenclature: in this chapter we have used the term "polyprenol" to mean a fully unsaturated polyprenyl alcohol. The number of isoprene units in the molecule is indicated following a dash. Thus, nonadecaprenol would be called polyprenol-19. Derivatives of polyprenoi are indicated, where appropriate, with the exception of the 2,3-dihydro derivative which is called "dolichol." "Prenol" is used to refer to both dolichol and polyprenol. 2 j. Burgos, F. W. Hemming, J. F. Pennock, and R. A. Morton, Biochem. J. 88, 470 (1963). R. K. Keller and W. L. Adair, Biochim. Biophys. Acta 489, 330 (1977). 4 R. W. Keenan and M. Krusczek, Anal. Biochem. 69, 504 (1975). W. L. Adair and S. Robertson, Biochem. J. 189, 441 (1980). 6 0 . Samuel, Z. Hachimi, and R. Azerad, Biochimie 56, 1279 (1974).
METHODS IN ENZYMOLOGY, VOL. 111
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182011-4
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to separate the two types of prenols by high-pressure liquid chromatography on silica, 7 a technique which is most effective when a single isoprenolog of each prenol is chromatographed. Should relatively large quantities of a given type of prenol be available, spectroscopic analysis (IR, NMR) can be successfully applied. 2,s,9 With regards to the chemistry of the monophosphate esters of polyprenol and dolichol, both are stable to treatment with strong alkali, allowing the use of saponification conditions during purification without serious losses. In contrast, mild acid degrades polyprenyl phosphate while dolichyl phosphate is stable to treatment even with strong acid.l° The glycosylated derivatives of dolichyl and polyprenyl phosphate are labile to alkali and acid. 1~Both treatments degrade all these compounds to the monophosphate form, with the important exception of dolichyl phosphomannose, which undergoes an elimination reaction to form mannose 2-phosphate and dolichoP 2 in the presence of base. Methods Thin-Layer Chromatography. Chromatography is performed on plastic-backed plates of silica gel 60 (0.25 mm thickness) from EM Labs. Tanks are equilibrated at least 1 hr before use. Detection of lipids is achieved by placing the plates in a TLC tank to which iodine crystals have been added. Alternatively, plates can be sprayed with anisaldehyde reagent and heated. High-Pressure Liquid Chromatography. An instrument capable of maintaining back pressures of 5000 psi is required. An ultraviolet monitor with variable wavelength detection is preferred; however, a fixed wavelength monitor at 214 nm is adequate. The monitor is connected to a stripchart recorder through an on-line integrator for quantitative analysis. We use a Laboratory Data Control Constametric IIG instrument with a Spectromonitor II variable wavelength detector, but any comparable instrument will suffice. For analysis of products from metabolic labeling studies, an on-line radioactivity monitor (e.g., Flow-One by Radiomatic Instruments) is convenient. Most commercial columns are adequate for the separations reported below; however we have found the Brownlee analytical cartridge columns to be especially useful. We use 5-/.~m, 22-cm columns to which are affixed 7 R. K. Keller, G. D. Rottler, and W. L. Adair, J. Chromatogr. 236, 230 (1982). s j. Feeney and F. W. Hemming, Anal. Biochem. 20, 1 (1967). 9 W. C. Breckenridge, L. S. Wolfe, and N. M, K. Ng Ying Kin, J. Neurochem. 21, 1311
(1973). i0 j. F. Wedgwood,C. D. Warren, and J. L. Strominger, J. Biol. Chem. 249, 63•6 (1974). 11C. D. Warren and R. W. Jeanloz, this series, Vol. 50 [8]. 12C. D. Warren, I. Y. Liu, A. Herscovics,and R. Jeanloz, J. Biol. Chem. 250, 8069(1975).
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3-cm guard cartridges in a single assembly. With this sytem, any significant loss of resolution or increase in back pressure can be easily remedied by insertion of a new guard cartridge. Solvents. All solvents are HPLC grade. "Reagent alcohol" is a specially prepared solvent for HPLC use from Fisher Scientific, The composition is ethanol/methanol/isopropanol (90/5/5). All solvent mixtures are passed through a filter-degasser (Lazar Research Labs) prior to use and stored in 4-liter bottles. Evaporations. Solvent removal is achieved using a 12-place evaporator (N-Evap, Organomation Associates) with the water bath set at 40° and argon or nitrogen as the flushing gas. Preparation of Labeled Compounds
Preparation of [3H]Polyprenol and [3H]Dolichol Principle. Dolichol or polyprenol is oxidized to the corresponding aldehyde by appropriate reagents. Reduction of the purified aldehyde product with NaB3H4 yields the desired labeled compounds. Procedures. The procedures described here are those reported by Adair et al. ~3and Adair and Robertson? In the oxidation of polyprenol, care must be taken to avoid cis-trans isomerization of the a-isoprene residue of the aldehyde product. Studies have shown that this side reaction is more prominent when using the chromium trioxide reagent and that it is enhanced by irradiation with UV light. 13 (Indeed, advantage can be taken of this side reaction as a convenient method to prepare the a-transpolyprenol from the naturally occurring cis compound.) A second problem is the occurrence of an appreciable amount of 1,4-addition when sodium borotritide is used,~4 thereby generating tritiated dolichol as a side product. Although the use of lithium aluminum hydride avoids 1,4-addition,14 high specific activity LiAI3H4 is not commercially available and the lability of this reagent makes it unattractive for routine laboratory use. [1-3H]Polyprenol. Polyprenol (2 mg) is dissolved in 100 txl hexane to which 10 mg MnO2 is added. After stirring 60 rain at 4 ° in a sealed, amber Reacti-vial (Pierce), the mixture is diluted with 2 ml hexane and centrifuged to remove the oxidizing reagent. Thin-layer chromatography of an aliquot of the supernatant on fluorescent dye-impregnated plates using chloroform as a solvent yields a single UV-positive spot (the allylic aldehyde) at R f = 0.8. Spraying with anisaldehyde reagent shows traces of unreacted polyprenol in addition to the major product polyprenal. If any z3 W. L. Adair, N. Cafmeyer, and R. K. Keller, J. Biol. Chem. 259, 4441 (1984). z4 M. R. Johnson and B. Rickborn, J. Org. Chem. 35, 1041 (1970).
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cis-trans isomerization occurs during workup the a-trans-polyprenal will appear as an additional UV-positive band migrating slightly slower than the ot-cis compound. Purification is effected by applying the mixture to a 1 × 3-cm column of alumina (Brockman grade 3, equilibrated with hexane) and eluting the polyprenal with hexane/ether (9/1). Fractions of 1 ml each are collected and aliquots monitored by thin-layer chromatography. After evaporation of the eluting solvent, the aldehyde is dissolved in 0.25 ml 1,2-dichloroethane and treated with 100 /~1 NaB3H4 (100 mCi, 2-4 Ci/ mmol) in alkaline ethanol (100/zl 1 N NaOH per 10 ml ethanol) for 90 rain at room temperature. The reaction is stopped with 10/xl concentrated acetic acid and applied to a 1 × 3-cm column of alumina as before, eluting the [l-3H]polyprenol with hexane/ether (4/1). Thin-layer chromatography in chloroform yields a single band with anisaldehyde spray and radiochromatogram scanning (Rf = 0.4), comigrating with authentic polyprenol (available from Sigma Chemical Co.). High-pressure liquid chromatography of such preparations, according to the method of Keller et al.,7 shows small amounts (generally less than 10%) of [3H]dolichol. [l-3H]Dolichol. Dolichol (2 rag) is dissolved in 80 /xl dry dichloromethane and stirred with 1 mg pyridinium chlorochromate. After 60 rain thin-layer chromatography (chloroform) indicates nearly quantitative conversion to a faster moving band (Rf = 0.8). Should the oxidation be incomplete, another addition of pyridium chlorochromate is made and the reaction stirred a further 30 min. The sample is diluted with 300 ~l dichloromethane and applied to a 1 × 5-cm column of Florisil equilibrated and eluted with dichloromethane. Fractions of about 1 ml are monitored by thin-layer chromatography; those containing the aldehyde product are pooled and evaporated to a small volume. Reduction with NaB3H4 and final purification of [ l-3H]dolichol follows the protocol outlined above for the preparation of [1-3H]polyprenol. Following purification, the radiolabeled samples are evaporated to dryness, dissolved in benzene, and transferred to 2-ml glass storage vials (Pierce). The vials are flushed with argon, capped with Teflon-lined septa, and stored in the refrigerator. The purity should be checked periodically by reversed phase HPLC. Over time, some degradation to polar derivatives has been observed to occur. Repurification can be achieved using Cl8 Sep-Pak columns (Waters Associates). The sample is applied in methanol/ethanol (75/25), rinsed with methanol/ethanol (50/50), and eluted with methanol/ethanol (25/75).
Preparation of Dolichyl [32p]Phosphate and Polyprenyl [uP]Phosphate Principle. The 3Zp-labeled phosphomonoesters of dolichol and polyprenol can be prepared chemically from the free alcohol and inorganic
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[32p]phosphate by a modification of the procedure of Cramer et al.,15 in which trichloroacetonitrile serves as the condensing agent. Procedure. In this procedure the prenol is reacted with a slight stoichiometric excess of inorganic [32P]phosphate in the presence of trichloroacetonitrile. This method has the advantage of producing only the monophosphate derivative and thus avoids the chromatographic purification protocol required in the procedure of Kandutsch et al) 6 Dolichyl [32P]phosphate can also be prepared chemically using [32P]POC13 by the method of Keenan. ~7 However, this procedure, while giving excellent yields, is limited by the relatively low specific activity of 32p product that has been attained (0.04 Ci/mol). In addition, since POCi3 cannot successfully phosphorylate allylic alcohols (W. L. Adair, unpublished data), the POCI3 method is not useful for preparing 3Zp-labeled polyprenyl phosphate. Dolichyl ff2p]Phosphate and Polyprenyl ff2p]Phosphate. Polyprenol or dolichol (1.0 mg, 0.77/~mol) is dissolved in 50/xl ethylene dichloride containing trichloroacetonitrile (2 t~mol). Bis(triethylammonium)[32plphosphate (1.0 txmole, 1.0 mCiY 8 in 50 txl acetonitrile is then added in a single portion. The reaction is then heated in a sealed vial at 50° for 1 hr. After cooling to room temperature, the reaction mixture is diluted with 2 ml chloroform, 1 ml methanol, and 0.75 ml water. Following centrifugation, the lower phase is applied to a 1 × 2-cm column of DEAE-cellulose (acetate form) equilibrated with chloroform/methanol (2/1). After rinsing with 10 ml chloroform/methanol (2/1), the product is eluted with 10 ml 0.3 M ammonium acetate in chloroform/methanol (2/1). One-quarter volume of water is added and the resulting lower phase, containing the labeled product, is collected after centrifugation. Thin-layer chromatography (chloroform/methanol/water, 65/35/4) of an aliquot of the washed eluate shows a single radioactive peak (Re = 0.4) comigrating with standard dolichyl phosphate. The average yield is about 3-4%, based on radioactivity. The final product is dissolved in chloroform/methanol (2/1), aliquoted into glass vials and stored under argon at - 7 0 ° .
Preparation of 3H-Labeled Dolichyl Phosphate Principle. In this method [3H]dolichol is reacted with POCI3 in the presence of triethylamine and the adduct hydrolyzed to produce the desired monophosphate product. ~5 F. Cramer, W. Rittersdorf, and W. Bohm, Chem. Ber. 654, 180 (1961). f6 A. A. Kandutsch, H. Paulus, E. Levine, and K. Bloch, J. Biol. Chem. 239, 2504 (1964). ~7R. W. Keenan, R. A. Martinez, and R. F. Williams, J. Biol. Chem. 257, 14817 (1982). z8 One micromole H3PO4 and 1 mCi H332PO4 (carrier free, HCI free) are taken to dryness in a 0.3-ml Reacti-vial. After an addition of 2/xmol triethylamine in 50/xl acetonitrile, the vial is capped and then vortexed well.
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Procedure. Although the procedure outlined above for the preparation of dolichyl [32P]phosphate can be employed for the preparation of [l-3H]dolichyl phosphate, a higher yield can be attained using a modification of the procedure of Danilov and Chojnacki ~9 which is described below. One drawback of this method is that it cannot be used to prepare tritiated polyprenyl phosphates: they must be prepared by other means, such as the procedure outlined above. [l-3H]Dolichyl Phosphate. [1-3H]Dolichol (106-109 dpm) is dissolved in 25 ~1 benzene and added to a solution containing 25/zl POCI3, 5 /zl triethylamine, and 25/zl benzene. After incubating 1 hr at room temperature, the reaction is stopped by the addition of 0.5 ml of a solution of acetone/water/triethylamine (88/10/2). The mixture is allowed to stand overnight (to complete the hydrolysis of the chlorophosphate derivative) and then evaporated to a small volume. To the residue is added 2 ml chloroform, 1 ml methanol, and 0.75 ml water. The lower phase obtained after centrifugation is washed twice with 50% aqueous methanol containing 1 M H3PO4 and taken to dryness. The phosphoric acid is included in the washes to facilitate extraction of the excess triethylamine, which if not removed will interfere with subsequent chromatographic steps. The washed product is dissolved in 3 ml chloroform/methanol (2/1) and purified by ion exchange chromatography as described above for the 32p_ labeled derivative. Overall yield is about 75%.
Isolation and Assays of Dolichols and Polyprenols The original procedure outlined by Burgos et al. z is used for the initial saponification and extraction of prenols. Various column techniques can then be employed for further purification prior to HPLC. These include chromatography on Florisil, 2° Lipidex, 2~ alumina, 2 or gel filtration using Fractogel 60003 (a polyvinyl acetate resin originally sold by Merck, but subsequently discontinued), and Sephadex LH-20. 4 Both adsorption and reversed-phase high-pressure liquid chromatography have been used successfully, the former having the advantage of yielding a single peak, the latter having the advantage of displaying the distribution of isoprenologs. The procedure which follows (Scheme l) is used routinely in our laboratory for the isolation and quantltation of dolichol and dolichyl phosphate from rat liver, but can be adapted to any tissue. ~9L. L. Danilov and T. Chojnacki, FEBS Lett. 131, 310 (1981). 2o K. K. Carroll, A. Vilim, and M. C. Woods, Lipids 8, 246 (1973). zl T. Mankowski, T. Jankowski, T. Chojnacki, and P. Franke, Biochemistry 15, 2125 (1976).
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Tissue
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flow through [ C18 Sep Pac
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HPLC SCHEME 1.
Isolation of Dolichol from Animal Tissues Procedure. To 1 g tissue (wet weight) in a 18 × 150-mm screw capped test tube is added 1 ml 60% KOH, 2 ml 0.25% pyrogallic acid in methanol, and l05 cpm of [3H]dolichol. The solution immediately turns dark brown in color. The tube is sealed with a Teflon-lined plastic cap and is placed in a boiling water bath for 1 hr, during which time complete dissolution of the tissue occurs. After incubation, the sample is cooled to room temperature and extracted three times with 3 ml diethyl ether. 22The extracts (total volume about 9 ml) are pooled in a 40-ml glass conical centrifuge tube with a Teflon-lined cap (Scientific Products C3985-40; Teflon lined cap is T1358-4) and washed once with an equal volume of 5% acetic acid. After centrifugation for 2 min at 1000 rpm, the upper phase is removed and taken to dryness. The residue, which sometimes contains small quantities (<100 /~l) of water is dissolved in 2 ml chloroform/methanol (2/1) and applied to a DEAE-cellulose column (2 ml packed resin in a 10-ml disposable polypropylene syringe, fitted with a precut Whatman GF/C filter to retain the resin). The column allows all dolichol, cholesterol, and other neutral lipids to pass through while retaining dolichyl phosphate) s The 2: Petroleum ether has also been used to extract saponification reactions. It has the advantage of not producing peroxides upon storage. Also, there may be fewer polar contaminants extracted into the upper phase. 23 This step can be omitted if quantitation of dolichol only is desired, since it is highly unlikely that any of the compounds which are retained by the DEAE-cellulose column copurify with dolichol.
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collected drop-through is treated with 0.25 volumes 0.88% KCI, vortexed, and centrifuged. The lower phase is evaporated to dryness and dissolved in 2 ml methanol. The sample is then applied to a C~8 Sep-Pak column (Waters) equilibrated in methanol. 24 The bulk of the sterols pass through the column unretarded while dolichol is retained. 25 After washing with 10 ml methanol, dolichol is eluted in good yield with 10 ml reagent alcohol. The alcohol fraction is then taken to dryness in preparation of the sample for HPLC analysis. The chief limitation of Sep-Pak columns is that sample size is generally restricted to less than 10 g wet weight of starting material. In those instances where large quantities of tissue are being processed for preparation of milligram quantities of prenols, purification can be achieved using large columns of alumina, 2 Florisil, z° or Lipidex 5000fl ~ the latter resin having the advantage of resolving isoprenolog species.
Isolation of Polyprenol from Plant Tissue Procedure. Although direct saponification of plant tissue followed by filtration and solvent extraction has been used by many laboratories, we have found it more convenient to extract the tissue prior to saponification. Thus, 2.5 g of chopped plant leaves is suspended in 15 ml acetone and pulverized with a Polytron homogenizer in a well-ventilated hood. The suspension is filtered and the residue and filtrate are retained. The residue is suspended in 20 ml petroleum ether (bp 30-50 °) and rehomogenized and filtered as before. Following a second petroleum ether extraction, the organic extracts are pooled and an appropriate radiolabeled internal standard (e.g., [3H]polyprenol-19) is added. The sample is then treated with 50 ml water. After vigorous shaking, the upper phase is collected and dried over anhydrous N a 2 S O 4 . The sample is then flash-evaporated and saponified by treatment with 10 ml of methanol/isopropanol/water (2/I/1), 8.0 g KOH, and 0.5 g pyrogallic acid in a boiling water bath. We routinely carry out the saponifications in a 40-ml screw-capped conical centrifuge tube. After 2 hr, the reaction is cooled to room temperature, diluted with 3 ml water, and extracted three times with 15 ml petroleum ether. The organic layers are pooled, backwashed with 50 ml water, and dried over Na2SO4. After flash-evaporation, the residue is taken up in 1 ml hexane and chromatographed on 1 g of alumina (Brockman grade 3), packed in a z4 Many investigators apply the total nonsaponifiable fraction directly to HPLC. However, we have observed that analysis of samples in this manner generally results in higher levels calculated for dolichol, indicating that contaminants comigrating with dolichol must be present. 25 T. K. Wong and W. J. Lennarz, J. Biol. Chem. 257, 6619 (1982).
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Pasteur pipet, and equilibrated with hexane. After application of the sample, the column is rinsed with hexane and then sequentially eluted with 5 and 8 ml portions of hexane/diethyl ether (4/1) and (2/1), respectively. Fractions of about 2 ml are collected and aliquots taken for analysis by scintillation counting or thin-layer chromatography (chloroform). Those fractions containing the bulk of the polyprenol are pooled, flash-evaporated, and dissolved in the appropriate solvent for analysis by HPLC. Prior purification using the Ci8 Sep-Pak method described above for dolichol is to be avoided in this case, since many plants contain prenols with less than 15 isoprene units. These are not effectively retained by the SepPak column, even when applied in various mixtures of methanol and water.
Assays of Total Dolichol and Polyprenol Assay by High-Pressure Liquid Chromatography. HPLC of dolichol and polyprenol, as mentioned above, can be carried out on silica or octadecyl-silica, depending on the information desired. For those instances in which direct quantitation as a single peak is preferred, we use silica HPLC with 0.3% reagent alcohol in hexane as a solvent. The dried sample is dissolved in 0.25 ml of the HPLC solvent, and 0.1 ml is drawn up into a 0. l-ml syringe. Any turbidity in the sample to be injected can usually be cleared by heating 30 sec in a 50° water bath. Fifty microliters is taken for liquid scintillation counting and the remaining 50 tA is injected into the HPLC. The elution position of dolichol and polyprenol is dependent on their chain length. 7 For polyprenoi-19 and dolichol-19, k' values of approximately 7 and 8, respectively, are obtained at a flow rate of I ml/min (Fig. la). Back pressures are on the order of 300 psi. Detection of prenols is by on-line UV monitoring at 210 nm. The lower limit of detection is about 10 ng using a high quality detector. By injecting fixed volumes (e.g., 50 txl using a 100 tzl sample loop) of different dilutions of the standardized prenol solution (see below), a standard curve of peak height (or area) vs micrograms prenol is prepared. The level of prenol in the original sample is then calculated from the following equation: /xg prenol g tissue
--
3H cpm prenol added/g 3H cpm injected
×
peak ht. of sample peak ht.//zg prenol
If the isoprene distribution of the prenol sample is desired, reversedphase chromatography is carried out. The system is analogous to that described above for silica, except that 5 tzm C~8 cartridges are used. The solvent system is isopropanol/methanol (1/1). At a flow rate of 1.0 ml/min,
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MINUTES FiG. 1. High-pressure liquid chromatography of long-chain prenols and prenyl phosphates. Adsorption chromatography on silica of (a) polyprenol-19 and dolichol-19 and (b) polyprenyl-19 phosphate and dolichyl-19 phosphate. (c) Reversed-phase chromatography of dolichyl phosphate from pig liver. Values above peaks refer to number of carbon residues in individual isoprenologs. Flow rate on all runs is 1.0 ml/min. Ultraviolet monitoring is at 210 nm. Solvent systems are described in text.
the isoprenologs elute within 20 min with baseline resolution, as shown in Fig. lc. Column back pressures are on the order of 2000-3000 psi. The above solvent system has proven convenient for separating isoprenologs containing 17-25 isoprene units. For shorter length species better resolution is obtained by decreasing the ratio of isopropanol to methanol. The isoprenolog peaks will appear as doublets if the sample (e.g., many plant tissues) contains both dolichols and polyprenols since these compounds are only partially resolved by reversed-phase HPLC. 26'z7 Confirmation of both types of prenols in the sample can be achieved by subjecting one of the isoprenolog doublets to adsorption HPLC, which yields baseline reso26 R. K. Keller, E. Jehle, and W. L. Adair, J. Biol. Chem. 257, 8985 (1982). 27 K. Ravi, J. W. Rip, and K. K. Carroll, Lipids 19, 401 (1984).
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lution of the two species, or by oxidation with MnO2, which selectively oxidizes only the a-unsaturated polyprenol. The purification procedure described above is generally satisfactory for the isolation of prenols in pure form from most tissues. If HPLC shows contaminants interfering with prenol analysis, then further purification is necessary. Samples are chromatographed on 1.5 × 30-cm columns of Sephadex LH-20 equilibrated in reagent alcohol. Prenols elute in approximately 25 ml and should be of sufficient purity for HPLC analysis. The analytical HPLC columns described above can handle samples containing up to 1 mg prenol. HPLC analysis of up to 5 mg prenols can be achieved using 10-cm-diameter preparative columns in either the adsorption (silica) or reversed-phase (C18) mode employing the solvents described above. Assay by Derivatization. The development of HPLC analysis has superceded the use of derivatization reactions for the analysis of dolichol and polyprenol. However, derivatization is still the method of choice for the standardization of prenol solutions to be used for HPLC analysis. The procedure which follows is from the original acetylation reaction described by Keller and Adair 3 and is based on the formation of a stable derivative using a reagent with known specific activity. [3H]Prenol is added in tracer quantities to determine the amount of starting material converted to product. To 0. l-ml Reacti-vials (Pierce) are added various estimated amounts of prenol (1-I0 ~g) and 300,000 dpm [3H]prenol. The samples are taken to dryness and 20/xl [14C]acetic anhydride (10/~Ci, 1/~mol) is added. The vials are capped with Teflon-lined septa and heated at 70° in a hood overnight. Slight loss of volume will be observed. After cooling, the samples are opened, evaporated to dryness in the hood, and dissolved in 1020/xl chloroform/methanol (2/1). Thin-layer chromatography is then carfled out in hexane/ether (8/2). Alongside the sample are spotted standards (5 txg each) of prenol and prenyl acetate (available from Sigma Chemical). After development, radioscanning shows three peaks: (1) at the origin, corresponding to excess unreacted 14C material, (2) at Rf = 0.2, corresponding to any unreacted [3H]prenol, and (3) at Rf = 0.7, corresponding to the double-labeled prenyl acetate. The latter two peaks should align with the corresponding standards. The prenyl acetate region is either subjected to sample oxidation or scraped from the plate into a 13 × 100mm disposable test tube and eluted with 5 ml diethyl ether. If the sample is eluted, the eluant is then transferred to a scintillation vial, dried, and counted for 3H and 14C in a detergent-containing scintillation fluid (e.g.,
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ACS, Amersham). The formula below is used to calculate the amount of prenol in the original sample. nanomoles prenol =
[3H]prenol added (dpm) 14C dpm in derivative 3H dpm in derivative x 14C acetic anhydride sp. act. (dprrgnmol)
The above calculation assumes that the mass of [3H]prenol is negligible. We routinely use specific activities in the range of 1-10 Ci/mmol, so that 300,000 dpm corresponds to about 14-140 pmol. Whether this is negligible in the above calculation depends on the amount of prenol derivatized. Correction for the mass of [3H]prenol is easily achieved by carrying out a derivatization reaction to which no unlabeled prenol is added. It should be noted that the absolute specific activity of the [3H]prenol can also be calculated from this reaction.
Isolation and Analysis o f Free Unesterified Prenol and Prenyl Fatty Acid Esters Procedure. Any of the common procedures using organic solvents can be employed for the initial extraction. These include the methods of Folch et a1.,28 Radin,29 and Bligh and Dyer. 3° To the organic extracts are added about 105 dpm each of [3H]prenol and [3H]prenyl palmitate (prepared from [3H]prenol and palmitoyl chloride3). Following any washing steps the sample can be chromatographed on Florisil for the separation of free and esterified prenol. 2° The free prenol fraction is saponified, purified, and assayed as described above. The ester fraction has been analyzed directly by reversed phase HPLC but cleaner preparations can be obtained if the sample is subjected to the saponification and purification procedure described above. Thus with the latter procedure, the prenyl esters will be analyzed as free prenol. Isolation and Assay of Dolichyl Phosphate Although the great majority of studies indicate that dolichyl phosphate is the prenyl phosphate involved in glycosyl transfer in eucaryotes, the possibility remains that the a-unsaturated polyprenyl phosphates may serve this function in some species, particularly plants and lower organisms. Accordingly, and because of the possibility that polyprenyl phosphate is a metabolic precursor for dolichyl phosphate, 3~it is desirable to J. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 226, 497 0957). 29 N. S. Radin, this series, Vol. 72 [1]. 30 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 0959). 3~ C. Vigo and W. L. Adair, Biosci. Rep. 2, 835 0982).
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have available procedures which allow isolation and assay of both of these long chain prenyl phosphates. Solvent systems have not yet been developed which allow resolution of polyprenyl phosphate and dolichyl phosphate by HPLC. It is therefore necessary to carry out indirect determinations. For example, advantage can be taken of the lability of polyprenyl phosphate to acid: analysis (e.g., by HPLC) of the acid-untreated and treated samples yields values for polyprenyl phosphate by difference. Alternatively, the prenyl phosphate fraction may be dephosphorylated using phosphatases. 32,33 The resulting product may then be subjected to silica HPLC, which resolves polyprenol from dolichol (see above). The disadvantage of this method is that it assumes that the phosphatases work equally well on dolichyl and polyprenyl phosphates. In theory, it should be possible to analyze for total dolichyl phosphate by carrying out extractions with chloroform/methanol (2/1) and chloroform/methanol/water (10/10/3) since all known dolichol-containing compounds should be soluble in one or both of these solvents. These extracts, when pooled and subjected to treatment with acid and base, should contain the total tissue dolichyl phosphate as free dolichyl phosphate. However, experiments in our laboratory have clearly demonstrated that the levels of dolichyl phosphate obtained from this procedure are significantly less than that obtained by direct saponification of the tissue. Studies have shown that the final insoluble residue after 10/10/3 extract contains a substantial amount of dolichyl phosphate (2-3/~g/g in rat liver). Although the basis for this finding is not yet apparent, it is clear that in order to carry out analysis of total dolichyl phosphate (minus dolichyl phosphomannose which is broken down to free dolicho112), tissue saponification and extraction are preferred to direct organic solvent extraction with subsequent acid-base treatments.
Isolation of Total Dolichyl Phosphate Procedure. The procedure described is for the analysis of dolichyl phosphate; if the sample is suspected of containing polyprenyl phosphate, it must be subjected to acid hydrolysis or enzymatic dephosphorylation (see above). The tissue is treated as described above for dolichol (see Scheme l). 32p. or 3H-labeled dolichyl phosphate is added to the saponification mixture to monitor purification and correct for yield. Following ether extraction and washing, the sample is dried, dissolved in chloroform/methanol (2/1), and applied to DEAE-cellulose as described above. The column is 32 D. D. Carson and W. J. Lennarz, J. Biol. Chem. 256, 4679 (1981). 33 H. Fujii, T. Koyama, and K. Ogura, Biochim. Biophys. Acta 712, 716 (1982).
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washed with I0 ml each of chloroform/methanol (2/I), chloroform/acetic acid (3/1), chloroform/methanol (2/1), and chloroform/methanol (2/1) containing 0.1 M ammonium acetate. The last fraction, containing the labeled dolichyl phosphate, is treated with 0.25 volumes water, vortexed, and centrifuged. The lower phase is underpipetted into a fresh tube and taken to dryness for subsequent derivatization or HPLC analysis (see below).
Assays of Dolichyl and Polyprenyl Phosphate Assay by High-Pressure Liquid Chromatography. HPLC of dolichyl phosphate, like dolichol, can be carried out in the adsorption or reversed phase modes. For adsorption chromatography on silica, we use the same column and conditions as described above for dolichol except that the solvent employed is hexane/isopropanol/1.4 M H3PO4 o r H2SO4 (965/35/ 0.5). A typical chromatographic run is shown in Fig. lb. Because of its highly apolar nature, dolichyl phosphate elutes early from this system (6 min) compared to other phospholipids. 34 Quantitation is achieved by preparing a curve generated from injection of standardized solutions (see below). If it is desired to use the HPLC-purified dolichyl phosphate in further studies (e.g., derivatization) in which the phosphoric acid would interfere, the sample can be treated with 1.5 volumes of isopropanol/ water (890/585). The resulting upper phase will contain all the dolichyl phosphate while the phosphoric acid quantitatively extracts into the lower phase. For the reversed-phase HPLC of dolichyl phosphate, the system developed by Chaudhary et al. 3sis employed. For preparation of solvent, 1 g crystalline H3PO4 (Fluka) is dissolved in 1 liter of isopropanol/methanol (1/1). The column and conditions are the same as for reversed-phase analysis of dolichol. As with dolichol, reversed-phase chromatography results in baseline separation of the isoprenolog species (Fig. lc). Assay by Derivatization. This procedure is based on that described by Keller et al.36 and involves the formation of a stable derivative of dolichyl phosphate with [~4C]phenvlchloroformate. O
O
O
O
II
I[
II
II
D o I - - O - - P - - O - + CI--C--O--C6H5 O-
~ DoI--O--P--O--C--O--C6Hs
O-
R. K. Keller, D. Armstrong, F. C. Crum, and N. Koppang, J. Neurochem. 42, 1040 (1984). 35 N. Chaudhary, D. J. Freeman, J. W. Rip, and K. K. Carroll, Lipids 17, 558 (1982). 36 R. K. Keller, J. W. Tamkun, and W. L. Adair, Biochemistry 20, 5831 (1981).
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DOLICHOL AND DOLICHYL PHOSPHATE
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(The preparation of the radiolabeled derivatizing reagent, a simple one-step procedure, is described in the original publication.) The purified dolichyl phosphate fraction (usually from DEAE-cellulose chromatography) is taken to dryness in a polypropylene centrifuge tube. To the residue is added 0.1 ml chloroform, 0.01 ml 7 mM triethylamine in chloroform, and 0.01 ml (I0 nmol, about 500,000 dpm) [~4C]phenyl chloroformate in hexane. The sample is taken to dryness at room temperature and dissolved in a small volume (10 ~1) of chloroform/methanol (2/1) and spotted on a silica thin-layer plate. The choice of developing solvent is based on the ambient humidity: with low relative humidity we use chloroform/methanol (7/1) while with high relative humidity acetone is employed. In a separate lane is spotted a standard, prepared from a reaction using unlabeled materials. Radiochromatogram scanning of the sample lane after development reveals a peak at the origin (unreacted dolichyl phosphate), a peak at Rr = 0.5 (the derivative), and two faster moving peaks near the solvent front which are derived from unreacted [14C]phenyl chloroformate. The derivative peak can either be subjected to sample oxidation 36 or scraped and eluted with 1.0 ml hexane/isopropanol/ water (2/6/1.5). From the 3H dpm (derived from the [3H]dolichyl phosphate added to the original sample) and the Jac dpm, the nanomoles of dolichyl phosphate in the sample is determined: (nmol Dol-P/g) =
3H dpm added/g ~4C dpm in derivative × 3H dpm in derivative [r4C]phenyl chloroformate sp. act. (dpm/nmol)
If it is deemed necessary to substract the mass of the [3H]dolichyl phosphate tracer added to the original sample, the procedure is analogous to that described above for dolichol derivatization. The [14C]phenyl chloroformate assay is rapid and allows more samples to be processed per unit time than HPLC analysis, since many derivatives can be applied to TLC plates in a single development. The sensitivity is comparable, since the specific activity of commercially available [14C]phenol (the starting material for the [14C]phenyl chloroformate synthesis) is on the order of 22,000-150,000 dpm/nmol. One possible disadvantage of derivatization is that the partially purified sample may contain contaminants which interfere with the phenyl chloroformate reaction. Such interference would indicate HPLC as the method of choice for quantitation of these samples.
DOLICHOL
AND DOL1CHYL
PHOSPHATE
201
[8] I s o l a t i o n a n d A s s a y o f Dolichol a n d D o l i c h y l P h o s p h a t e
By W. LEE ADAm and R. KENNEDY KELLER The isolation and characterization of long-chain prenols t and their monophosphate derivatives are complicated by several factors. First, they are present in small quantities in most tissues examined (5-100/zg/g wet weight). As a result, precise analysis requires the inclusion of a radioactive tracer to monitor purification and correct for losses. Second, the prenols occur naturally in a variety of derivatized states, including acyl and phosphomonoesters of the free alcohols as well as the many glycosylated forms involved in heteroglycan biosynthesis. Since simultaneous analysis of all these derivatives would be a complex task, it is convenient to convert them to a few easily assayable species, namely the free alcohol and the monophosphate ester. For animal tissues this is generally carried out by direct saponification, 2 which completely dissolves the tissue and converts all the dolichol derivatives to dolichol and dolichyl phosphate. With plant tissues (which often contain large amounts of cellulose), it is preferable to extract the prenols with organic solvents prior to saponification. Once extracted, the purification and assay of the prenols and prenyl phosphates have proved a challenge for chromatographers and chemists alike. The chemistry of the long chain prenols is typical for that of allylic and saturated primary alcohols with regards to the hydroxyl end of the molecule. That is, the prenols may be reacted with acyl anhydrides and chlorides to form esters. 3 In addition, they can undergo oxidation to form prenals with a variety of oxidants, such as chromium trioxide-pyridine,4 pyridinium chlorochromate, 5 and manganese dioxide.6 It should be noted that treatment with manganese dioxide forms the basis of a convenient method to distinguish between polyprenol and dolichol, since only the former compound is susceptible to oxidation. An alternative procedure is 1 Nomenclature: in this chapter we have used the term "polyprenol" to mean a fully unsaturated polyprenyl alcohol. The number of isoprene units in the molecule is indicated following a dash. Thus, nonadecaprenol would be called polyprenol-19. Derivatives of polyprenoi are indicated, where appropriate, with the exception of the 2,3-dihydro derivative which is called "dolichol." "Prenol" is used to refer to both dolichol and polyprenol. 2 j. Burgos, F. W. Hemming, J. F. Pennock, and R. A. Morton, Biochem. J. 88, 470 (1963). R. K. Keller and W. L. Adair, Biochim. Biophys. Acta 489, 330 (1977). 4 R. W. Keenan and M. Krusczek, Anal. Biochem. 69, 504 (1975). W. L. Adair and S. Robertson, Biochem. J. 189, 441 (1980). 6 0 . Samuel, Z. Hachimi, and R. Azerad, Biochimie 56, 1279 (1974).
METHODS IN ENZYMOLOGY, VOL. 111
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182011-4
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to separate the two types of prenols by high-pressure liquid chromatography on silica, 7 a technique which is most effective when a single isoprenolog of each prenol is chromatographed. Should relatively large quantities of a given type of prenol be available, spectroscopic analysis (IR, NMR) can be successfully applied. 2,s,9 With regards to the chemistry of the monophosphate esters of polyprenol and dolichol, both are stable to treatment with strong alkali, allowing the use of saponification conditions during purification without serious losses. In contrast, mild acid degrades polyprenyl phosphate while dolichyl phosphate is stable to treatment even with strong acid.l° The glycosylated derivatives of dolichyl and polyprenyl phosphate are labile to alkali and acid. 1~Both treatments degrade all these compounds to the monophosphate form, with the important exception of dolichyl phosphomannose, which undergoes an elimination reaction to form mannose 2-phosphate and dolichoP 2 in the presence of base. Methods Thin-Layer Chromatography. Chromatography is performed on plastic-backed plates of silica gel 60 (0.25 mm thickness) from EM Labs. Tanks are equilibrated at least 1 hr before use. Detection of lipids is achieved by placing the plates in a TLC tank to which iodine crystals have been added. Alternatively, plates can be sprayed with anisaldehyde reagent and heated. High-Pressure Liquid Chromatography. An instrument capable of maintaining back pressures of 5000 psi is required. An ultraviolet monitor with variable wavelength detection is preferred; however, a fixed wavelength monitor at 214 nm is adequate. The monitor is connected to a stripchart recorder through an on-line integrator for quantitative analysis. We use a Laboratory Data Control Constametric IIG instrument with a Spectromonitor II variable wavelength detector, but any comparable instrument will suffice. For analysis of products from metabolic labeling studies, an on-line radioactivity monitor (e.g., Flow-One by Radiomatic Instruments) is convenient. Most commercial columns are adequate for the separations reported below; however we have found the Brownlee analytical cartridge columns to be especially useful. We use 5-/.~m, 22-cm columns to which are affixed 7 R. K. Keller, G. D. Rottler, and W. L. Adair, J. Chromatogr. 236, 230 (1982). s j. Feeney and F. W. Hemming, Anal. Biochem. 20, 1 (1967). 9 W. C. Breckenridge, L. S. Wolfe, and N. M, K. Ng Ying Kin, J. Neurochem. 21, 1311
(1973). i0 j. F. Wedgwood,C. D. Warren, and J. L. Strominger, J. Biol. Chem. 249, 63•6 (1974). 11C. D. Warren and R. W. Jeanloz, this series, Vol. 50 [8]. 12C. D. Warren, I. Y. Liu, A. Herscovics,and R. Jeanloz, J. Biol. Chem. 250, 8069(1975).
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DOLICHOL AND DOLICHYL PHOSPHATE
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3-cm guard cartridges in a single assembly. With this sytem, any significant loss of resolution or increase in back pressure can be easily remedied by insertion of a new guard cartridge. Solvents. All solvents are HPLC grade. "Reagent alcohol" is a specially prepared solvent for HPLC use from Fisher Scientific, The composition is ethanol/methanol/isopropanol (90/5/5). All solvent mixtures are passed through a filter-degasser (Lazar Research Labs) prior to use and stored in 4-liter bottles. Evaporations. Solvent removal is achieved using a 12-place evaporator (N-Evap, Organomation Associates) with the water bath set at 40° and argon or nitrogen as the flushing gas. Preparation of Labeled Compounds
Preparation of [3H]Polyprenol and [3H]Dolichol Principle. Dolichol or polyprenol is oxidized to the corresponding aldehyde by appropriate reagents. Reduction of the purified aldehyde product with NaB3H4 yields the desired labeled compounds. Procedures. The procedures described here are those reported by Adair et al. ~3and Adair and Robertson? In the oxidation of polyprenol, care must be taken to avoid cis-trans isomerization of the a-isoprene residue of the aldehyde product. Studies have shown that this side reaction is more prominent when using the chromium trioxide reagent and that it is enhanced by irradiation with UV light. 13 (Indeed, advantage can be taken of this side reaction as a convenient method to prepare the a-transpolyprenol from the naturally occurring cis compound.) A second problem is the occurrence of an appreciable amount of 1,4-addition when sodium borotritide is used,~4 thereby generating tritiated dolichol as a side product. Although the use of lithium aluminum hydride avoids 1,4-addition,14 high specific activity LiAI3H4 is not commercially available and the lability of this reagent makes it unattractive for routine laboratory use. [1-3H]Polyprenol. Polyprenol (2 mg) is dissolved in 100 txl hexane to which 10 mg MnO2 is added. After stirring 60 rain at 4 ° in a sealed, amber Reacti-vial (Pierce), the mixture is diluted with 2 ml hexane and centrifuged to remove the oxidizing reagent. Thin-layer chromatography of an aliquot of the supernatant on fluorescent dye-impregnated plates using chloroform as a solvent yields a single UV-positive spot (the allylic aldehyde) at R f = 0.8. Spraying with anisaldehyde reagent shows traces of unreacted polyprenol in addition to the major product polyprenal. If any z3 W. L. Adair, N. Cafmeyer, and R. K. Keller, J. Biol. Chem. 259, 4441 (1984). z4 M. R. Johnson and B. Rickborn, J. Org. Chem. 35, 1041 (1970).
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cis-trans isomerization occurs during workup the a-trans-polyprenal will appear as an additional UV-positive band migrating slightly slower than the ot-cis compound. Purification is effected by applying the mixture to a 1 × 3-cm column of alumina (Brockman grade 3, equilibrated with hexane) and eluting the polyprenal with hexane/ether (9/1). Fractions of 1 ml each are collected and aliquots monitored by thin-layer chromatography. After evaporation of the eluting solvent, the aldehyde is dissolved in 0.25 ml 1,2-dichloroethane and treated with 100 /~1 NaB3H4 (100 mCi, 2-4 Ci/ mmol) in alkaline ethanol (100/zl 1 N NaOH per 10 ml ethanol) for 90 rain at room temperature. The reaction is stopped with 10/xl concentrated acetic acid and applied to a 1 × 3-cm column of alumina as before, eluting the [l-3H]polyprenol with hexane/ether (4/1). Thin-layer chromatography in chloroform yields a single band with anisaldehyde spray and radiochromatogram scanning (Rf = 0.4), comigrating with authentic polyprenol (available from Sigma Chemical Co.). High-pressure liquid chromatography of such preparations, according to the method of Keller et al.,7 shows small amounts (generally less than 10%) of [3H]dolichol. [l-3H]Dolichol. Dolichol (2 rag) is dissolved in 80 /xl dry dichloromethane and stirred with 1 mg pyridinium chlorochromate. After 60 rain thin-layer chromatography (chloroform) indicates nearly quantitative conversion to a faster moving band (Rf = 0.8). Should the oxidation be incomplete, another addition of pyridium chlorochromate is made and the reaction stirred a further 30 min. The sample is diluted with 300 ~l dichloromethane and applied to a 1 × 5-cm column of Florisil equilibrated and eluted with dichloromethane. Fractions of about 1 ml are monitored by thin-layer chromatography; those containing the aldehyde product are pooled and evaporated to a small volume. Reduction with NaB3H4 and final purification of [ l-3H]dolichol follows the protocol outlined above for the preparation of [1-3H]polyprenol. Following purification, the radiolabeled samples are evaporated to dryness, dissolved in benzene, and transferred to 2-ml glass storage vials (Pierce). The vials are flushed with argon, capped with Teflon-lined septa, and stored in the refrigerator. The purity should be checked periodically by reversed phase HPLC. Over time, some degradation to polar derivatives has been observed to occur. Repurification can be achieved using Cl8 Sep-Pak columns (Waters Associates). The sample is applied in methanol/ethanol (75/25), rinsed with methanol/ethanol (50/50), and eluted with methanol/ethanol (25/75).
Preparation of Dolichyl [32p]Phosphate and Polyprenyl [uP]Phosphate Principle. The 3Zp-labeled phosphomonoesters of dolichol and polyprenol can be prepared chemically from the free alcohol and inorganic
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DOLICHOL AND DOLICHYL PHOSPHATE
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[32p]phosphate by a modification of the procedure of Cramer et al.,15 in which trichloroacetonitrile serves as the condensing agent. Procedure. In this procedure the prenol is reacted with a slight stoichiometric excess of inorganic [32P]phosphate in the presence of trichloroacetonitrile. This method has the advantage of producing only the monophosphate derivative and thus avoids the chromatographic purification protocol required in the procedure of Kandutsch et al) 6 Dolichyl [32P]phosphate can also be prepared chemically using [32P]POC13 by the method of Keenan. ~7 However, this procedure, while giving excellent yields, is limited by the relatively low specific activity of 32p product that has been attained (0.04 Ci/mol). In addition, since POCi3 cannot successfully phosphorylate allylic alcohols (W. L. Adair, unpublished data), the POCI3 method is not useful for preparing 3Zp-labeled polyprenyl phosphate. Dolichyl ff2p]Phosphate and Polyprenyl ff2p]Phosphate. Polyprenol or dolichol (1.0 mg, 0.77/~mol) is dissolved in 50/xl ethylene dichloride containing trichloroacetonitrile (2 t~mol). Bis(triethylammonium)[32plphosphate (1.0 txmole, 1.0 mCiY 8 in 50 txl acetonitrile is then added in a single portion. The reaction is then heated in a sealed vial at 50° for 1 hr. After cooling to room temperature, the reaction mixture is diluted with 2 ml chloroform, 1 ml methanol, and 0.75 ml water. Following centrifugation, the lower phase is applied to a 1 × 2-cm column of DEAE-cellulose (acetate form) equilibrated with chloroform/methanol (2/1). After rinsing with 10 ml chloroform/methanol (2/1), the product is eluted with 10 ml 0.3 M ammonium acetate in chloroform/methanol (2/1). One-quarter volume of water is added and the resulting lower phase, containing the labeled product, is collected after centrifugation. Thin-layer chromatography (chloroform/methanol/water, 65/35/4) of an aliquot of the washed eluate shows a single radioactive peak (Re = 0.4) comigrating with standard dolichyl phosphate. The average yield is about 3-4%, based on radioactivity. The final product is dissolved in chloroform/methanol (2/1), aliquoted into glass vials and stored under argon at - 7 0 ° .
Preparation of 3H-Labeled Dolichyl Phosphate Principle. In this method [3H]dolichol is reacted with POCI3 in the presence of triethylamine and the adduct hydrolyzed to produce the desired monophosphate product. ~5 F. Cramer, W. Rittersdorf, and W. Bohm, Chem. Ber. 654, 180 (1961). f6 A. A. Kandutsch, H. Paulus, E. Levine, and K. Bloch, J. Biol. Chem. 239, 2504 (1964). ~7R. W. Keenan, R. A. Martinez, and R. F. Williams, J. Biol. Chem. 257, 14817 (1982). z8 One micromole H3PO4 and 1 mCi H332PO4 (carrier free, HCI free) are taken to dryness in a 0.3-ml Reacti-vial. After an addition of 2/xmol triethylamine in 50/xl acetonitrile, the vial is capped and then vortexed well.
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METHODOLOGY
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Procedure. Although the procedure outlined above for the preparation of dolichyl [32P]phosphate can be employed for the preparation of [l-3H]dolichyl phosphate, a higher yield can be attained using a modification of the procedure of Danilov and Chojnacki ~9 which is described below. One drawback of this method is that it cannot be used to prepare tritiated polyprenyl phosphates: they must be prepared by other means, such as the procedure outlined above. [l-3H]Dolichyl Phosphate. [1-3H]Dolichol (106-109 dpm) is dissolved in 25 ~1 benzene and added to a solution containing 25/zl POCI3, 5 /zl triethylamine, and 25/zl benzene. After incubating 1 hr at room temperature, the reaction is stopped by the addition of 0.5 ml of a solution of acetone/water/triethylamine (88/10/2). The mixture is allowed to stand overnight (to complete the hydrolysis of the chlorophosphate derivative) and then evaporated to a small volume. To the residue is added 2 ml chloroform, 1 ml methanol, and 0.75 ml water. The lower phase obtained after centrifugation is washed twice with 50% aqueous methanol containing 1 M H3PO4 and taken to dryness. The phosphoric acid is included in the washes to facilitate extraction of the excess triethylamine, which if not removed will interfere with subsequent chromatographic steps. The washed product is dissolved in 3 ml chloroform/methanol (2/1) and purified by ion exchange chromatography as described above for the 32p_ labeled derivative. Overall yield is about 75%.
Isolation and Assays of Dolichols and Polyprenols The original procedure outlined by Burgos et al. z is used for the initial saponification and extraction of prenols. Various column techniques can then be employed for further purification prior to HPLC. These include chromatography on Florisil, 2° Lipidex, 2~ alumina, 2 or gel filtration using Fractogel 60003 (a polyvinyl acetate resin originally sold by Merck, but subsequently discontinued), and Sephadex LH-20. 4 Both adsorption and reversed-phase high-pressure liquid chromatography have been used successfully, the former having the advantage of yielding a single peak, the latter having the advantage of displaying the distribution of isoprenologs. The procedure which follows (Scheme l) is used routinely in our laboratory for the isolation and quantltation of dolichol and dolichyl phosphate from rat liver, but can be adapted to any tissue. ~9L. L. Danilov and T. Chojnacki, FEBS Lett. 131, 310 (1981). 2o K. K. Carroll, A. Vilim, and M. C. Woods, Lipids 8, 246 (1973). zl T. Mankowski, T. Jankowski, T. Chojnacki, and P. Franke, Biochemistry 15, 2125 (1976).
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DOLICHOL AND DOLICHYL PHOSPHATE
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Tissue
I I I
Saponify Ether Extract DEAE Cellulose in C/M
I
I
I
flow through [ C18 Sep Pac
I
i
NH4CHaCOOeluate (Prenyl Phosphates)
I
I. M e O H
2. E t O H
[Sterols]
[Prenols)
I
HPLC
I
HPLC
I
HPLC SCHEME 1.
Isolation of Dolichol from Animal Tissues Procedure. To 1 g tissue (wet weight) in a 18 × 150-mm screw capped test tube is added 1 ml 60% KOH, 2 ml 0.25% pyrogallic acid in methanol, and l05 cpm of [3H]dolichol. The solution immediately turns dark brown in color. The tube is sealed with a Teflon-lined plastic cap and is placed in a boiling water bath for 1 hr, during which time complete dissolution of the tissue occurs. After incubation, the sample is cooled to room temperature and extracted three times with 3 ml diethyl ether. 22The extracts (total volume about 9 ml) are pooled in a 40-ml glass conical centrifuge tube with a Teflon-lined cap (Scientific Products C3985-40; Teflon lined cap is T1358-4) and washed once with an equal volume of 5% acetic acid. After centrifugation for 2 min at 1000 rpm, the upper phase is removed and taken to dryness. The residue, which sometimes contains small quantities (<100 /~l) of water is dissolved in 2 ml chloroform/methanol (2/1) and applied to a DEAE-cellulose column (2 ml packed resin in a 10-ml disposable polypropylene syringe, fitted with a precut Whatman GF/C filter to retain the resin). The column allows all dolichol, cholesterol, and other neutral lipids to pass through while retaining dolichyl phosphate) s The 2: Petroleum ether has also been used to extract saponification reactions. It has the advantage of not producing peroxides upon storage. Also, there may be fewer polar contaminants extracted into the upper phase. 23 This step can be omitted if quantitation of dolichol only is desired, since it is highly unlikely that any of the compounds which are retained by the DEAE-cellulose column copurify with dolichol.
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MEXnODOLOGY
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collected drop-through is treated with 0.25 volumes 0.88% KCI, vortexed, and centrifuged. The lower phase is evaporated to dryness and dissolved in 2 ml methanol. The sample is then applied to a C~8 Sep-Pak column (Waters) equilibrated in methanol. 24 The bulk of the sterols pass through the column unretarded while dolichol is retained. 25 After washing with 10 ml methanol, dolichol is eluted in good yield with 10 ml reagent alcohol. The alcohol fraction is then taken to dryness in preparation of the sample for HPLC analysis. The chief limitation of Sep-Pak columns is that sample size is generally restricted to less than 10 g wet weight of starting material. In those instances where large quantities of tissue are being processed for preparation of milligram quantities of prenols, purification can be achieved using large columns of alumina, 2 Florisil, z° or Lipidex 5000fl ~ the latter resin having the advantage of resolving isoprenolog species.
Isolation of Polyprenol from Plant Tissue Procedure. Although direct saponification of plant tissue followed by filtration and solvent extraction has been used by many laboratories, we have found it more convenient to extract the tissue prior to saponification. Thus, 2.5 g of chopped plant leaves is suspended in 15 ml acetone and pulverized with a Polytron homogenizer in a well-ventilated hood. The suspension is filtered and the residue and filtrate are retained. The residue is suspended in 20 ml petroleum ether (bp 30-50 °) and rehomogenized and filtered as before. Following a second petroleum ether extraction, the organic extracts are pooled and an appropriate radiolabeled internal standard (e.g., [3H]polyprenol-19) is added. The sample is then treated with 50 ml water. After vigorous shaking, the upper phase is collected and dried over anhydrous N a 2 S O 4 . The sample is then flash-evaporated and saponified by treatment with 10 ml of methanol/isopropanol/water (2/I/1), 8.0 g KOH, and 0.5 g pyrogallic acid in a boiling water bath. We routinely carry out the saponifications in a 40-ml screw-capped conical centrifuge tube. After 2 hr, the reaction is cooled to room temperature, diluted with 3 ml water, and extracted three times with 15 ml petroleum ether. The organic layers are pooled, backwashed with 50 ml water, and dried over Na2SO4. After flash-evaporation, the residue is taken up in 1 ml hexane and chromatographed on 1 g of alumina (Brockman grade 3), packed in a z4 Many investigators apply the total nonsaponifiable fraction directly to HPLC. However, we have observed that analysis of samples in this manner generally results in higher levels calculated for dolichol, indicating that contaminants comigrating with dolichol must be present. 25 T. K. Wong and W. J. Lennarz, J. Biol. Chem. 257, 6619 (1982).
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DOLICHOL AND DOLICHYL PHOSPHATE
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Pasteur pipet, and equilibrated with hexane. After application of the sample, the column is rinsed with hexane and then sequentially eluted with 5 and 8 ml portions of hexane/diethyl ether (4/1) and (2/1), respectively. Fractions of about 2 ml are collected and aliquots taken for analysis by scintillation counting or thin-layer chromatography (chloroform). Those fractions containing the bulk of the polyprenol are pooled, flash-evaporated, and dissolved in the appropriate solvent for analysis by HPLC. Prior purification using the Ci8 Sep-Pak method described above for dolichol is to be avoided in this case, since many plants contain prenols with less than 15 isoprene units. These are not effectively retained by the SepPak column, even when applied in various mixtures of methanol and water.
Assays of Total Dolichol and Polyprenol Assay by High-Pressure Liquid Chromatography. HPLC of dolichol and polyprenol, as mentioned above, can be carried out on silica or octadecyl-silica, depending on the information desired. For those instances in which direct quantitation as a single peak is preferred, we use silica HPLC with 0.3% reagent alcohol in hexane as a solvent. The dried sample is dissolved in 0.25 ml of the HPLC solvent, and 0.1 ml is drawn up into a 0. l-ml syringe. Any turbidity in the sample to be injected can usually be cleared by heating 30 sec in a 50° water bath. Fifty microliters is taken for liquid scintillation counting and the remaining 50 tA is injected into the HPLC. The elution position of dolichol and polyprenol is dependent on their chain length. 7 For polyprenoi-19 and dolichol-19, k' values of approximately 7 and 8, respectively, are obtained at a flow rate of I ml/min (Fig. la). Back pressures are on the order of 300 psi. Detection of prenols is by on-line UV monitoring at 210 nm. The lower limit of detection is about 10 ng using a high quality detector. By injecting fixed volumes (e.g., 50 txl using a 100 tzl sample loop) of different dilutions of the standardized prenol solution (see below), a standard curve of peak height (or area) vs micrograms prenol is prepared. The level of prenol in the original sample is then calculated from the following equation: /xg prenol g tissue
--
3H cpm prenol added/g 3H cpm injected
×
peak ht. of sample peak ht.//zg prenol
If the isoprene distribution of the prenol sample is desired, reversedphase chromatography is carried out. The system is analogous to that described above for silica, except that 5 tzm C~8 cartridges are used. The solvent system is isopropanol/methanol (1/1). At a flow rate of 1.0 ml/min,
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METHODOLOGY
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i
b
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MINUTES FiG. 1. High-pressure liquid chromatography of long-chain prenols and prenyl phosphates. Adsorption chromatography on silica of (a) polyprenol-19 and dolichol-19 and (b) polyprenyl-19 phosphate and dolichyl-19 phosphate. (c) Reversed-phase chromatography of dolichyl phosphate from pig liver. Values above peaks refer to number of carbon residues in individual isoprenologs. Flow rate on all runs is 1.0 ml/min. Ultraviolet monitoring is at 210 nm. Solvent systems are described in text.
the isoprenologs elute within 20 min with baseline resolution, as shown in Fig. lc. Column back pressures are on the order of 2000-3000 psi. The above solvent system has proven convenient for separating isoprenologs containing 17-25 isoprene units. For shorter length species better resolution is obtained by decreasing the ratio of isopropanol to methanol. The isoprenolog peaks will appear as doublets if the sample (e.g., many plant tissues) contains both dolichols and polyprenols since these compounds are only partially resolved by reversed-phase HPLC. 26'z7 Confirmation of both types of prenols in the sample can be achieved by subjecting one of the isoprenolog doublets to adsorption HPLC, which yields baseline reso26 R. K. Keller, E. Jehle, and W. L. Adair, J. Biol. Chem. 257, 8985 (1982). 27 K. Ravi, J. W. Rip, and K. K. Carroll, Lipids 19, 401 (1984).
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DOLICHOL AND DOLICHYL PHOSPHATE
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lution of the two species, or by oxidation with MnO2, which selectively oxidizes only the a-unsaturated polyprenol. The purification procedure described above is generally satisfactory for the isolation of prenols in pure form from most tissues. If HPLC shows contaminants interfering with prenol analysis, then further purification is necessary. Samples are chromatographed on 1.5 × 30-cm columns of Sephadex LH-20 equilibrated in reagent alcohol. Prenols elute in approximately 25 ml and should be of sufficient purity for HPLC analysis. The analytical HPLC columns described above can handle samples containing up to 1 mg prenol. HPLC analysis of up to 5 mg prenols can be achieved using 10-cm-diameter preparative columns in either the adsorption (silica) or reversed-phase (C18) mode employing the solvents described above. Assay by Derivatization. The development of HPLC analysis has superceded the use of derivatization reactions for the analysis of dolichol and polyprenol. However, derivatization is still the method of choice for the standardization of prenol solutions to be used for HPLC analysis. The procedure which follows is from the original acetylation reaction described by Keller and Adair 3 and is based on the formation of a stable derivative using a reagent with known specific activity. [3H]Prenol is added in tracer quantities to determine the amount of starting material converted to product. To 0. l-ml Reacti-vials (Pierce) are added various estimated amounts of prenol (1-I0 ~g) and 300,000 dpm [3H]prenol. The samples are taken to dryness and 20/xl [14C]acetic anhydride (10/~Ci, 1/~mol) is added. The vials are capped with Teflon-lined septa and heated at 70° in a hood overnight. Slight loss of volume will be observed. After cooling, the samples are opened, evaporated to dryness in the hood, and dissolved in 1020/xl chloroform/methanol (2/1). Thin-layer chromatography is then carfled out in hexane/ether (8/2). Alongside the sample are spotted standards (5 txg each) of prenol and prenyl acetate (available from Sigma Chemical). After development, radioscanning shows three peaks: (1) at the origin, corresponding to excess unreacted 14C material, (2) at Rf = 0.2, corresponding to any unreacted [3H]prenol, and (3) at Rf = 0.7, corresponding to the double-labeled prenyl acetate. The latter two peaks should align with the corresponding standards. The prenyl acetate region is either subjected to sample oxidation or scraped from the plate into a 13 × 100mm disposable test tube and eluted with 5 ml diethyl ether. If the sample is eluted, the eluant is then transferred to a scintillation vial, dried, and counted for 3H and 14C in a detergent-containing scintillation fluid (e.g.,
212
METHODOLOGY
[8]
ACS, Amersham). The formula below is used to calculate the amount of prenol in the original sample. nanomoles prenol =
[3H]prenol added (dpm) 14C dpm in derivative 3H dpm in derivative x 14C acetic anhydride sp. act. (dprrgnmol)
The above calculation assumes that the mass of [3H]prenol is negligible. We routinely use specific activities in the range of 1-10 Ci/mmol, so that 300,000 dpm corresponds to about 14-140 pmol. Whether this is negligible in the above calculation depends on the amount of prenol derivatized. Correction for the mass of [3H]prenol is easily achieved by carrying out a derivatization reaction to which no unlabeled prenol is added. It should be noted that the absolute specific activity of the [3H]prenol can also be calculated from this reaction.
Isolation and Analysis o f Free Unesterified Prenol and Prenyl Fatty Acid Esters Procedure. Any of the common procedures using organic solvents can be employed for the initial extraction. These include the methods of Folch et a1.,28 Radin,29 and Bligh and Dyer. 3° To the organic extracts are added about 105 dpm each of [3H]prenol and [3H]prenyl palmitate (prepared from [3H]prenol and palmitoyl chloride3). Following any washing steps the sample can be chromatographed on Florisil for the separation of free and esterified prenol. 2° The free prenol fraction is saponified, purified, and assayed as described above. The ester fraction has been analyzed directly by reversed phase HPLC but cleaner preparations can be obtained if the sample is subjected to the saponification and purification procedure described above. Thus with the latter procedure, the prenyl esters will be analyzed as free prenol. Isolation and Assay of Dolichyl Phosphate Although the great majority of studies indicate that dolichyl phosphate is the prenyl phosphate involved in glycosyl transfer in eucaryotes, the possibility remains that the a-unsaturated polyprenyl phosphates may serve this function in some species, particularly plants and lower organisms. Accordingly, and because of the possibility that polyprenyl phosphate is a metabolic precursor for dolichyl phosphate, 3~it is desirable to J. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 226, 497 0957). 29 N. S. Radin, this series, Vol. 72 [1]. 30 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 0959). 3~ C. Vigo and W. L. Adair, Biosci. Rep. 2, 835 0982).
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DOLICHOL AND DOLICHYL PHOSPHATE
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have available procedures which allow isolation and assay of both of these long chain prenyl phosphates. Solvent systems have not yet been developed which allow resolution of polyprenyl phosphate and dolichyl phosphate by HPLC. It is therefore necessary to carry out indirect determinations. For example, advantage can be taken of the lability of polyprenyl phosphate to acid: analysis (e.g., by HPLC) of the acid-untreated and treated samples yields values for polyprenyl phosphate by difference. Alternatively, the prenyl phosphate fraction may be dephosphorylated using phosphatases. 32,33 The resulting product may then be subjected to silica HPLC, which resolves polyprenol from dolichol (see above). The disadvantage of this method is that it assumes that the phosphatases work equally well on dolichyl and polyprenyl phosphates. In theory, it should be possible to analyze for total dolichyl phosphate by carrying out extractions with chloroform/methanol (2/1) and chloroform/methanol/water (10/10/3) since all known dolichol-containing compounds should be soluble in one or both of these solvents. These extracts, when pooled and subjected to treatment with acid and base, should contain the total tissue dolichyl phosphate as free dolichyl phosphate. However, experiments in our laboratory have clearly demonstrated that the levels of dolichyl phosphate obtained from this procedure are significantly less than that obtained by direct saponification of the tissue. Studies have shown that the final insoluble residue after 10/10/3 extract contains a substantial amount of dolichyl phosphate (2-3/~g/g in rat liver). Although the basis for this finding is not yet apparent, it is clear that in order to carry out analysis of total dolichyl phosphate (minus dolichyl phosphomannose which is broken down to free dolicho112), tissue saponification and extraction are preferred to direct organic solvent extraction with subsequent acid-base treatments.
Isolation of Total Dolichyl Phosphate Procedure. The procedure described is for the analysis of dolichyl phosphate; if the sample is suspected of containing polyprenyl phosphate, it must be subjected to acid hydrolysis or enzymatic dephosphorylation (see above). The tissue is treated as described above for dolichol (see Scheme l). 32p. or 3H-labeled dolichyl phosphate is added to the saponification mixture to monitor purification and correct for yield. Following ether extraction and washing, the sample is dried, dissolved in chloroform/methanol (2/1), and applied to DEAE-cellulose as described above. The column is 32 D. D. Carson and W. J. Lennarz, J. Biol. Chem. 256, 4679 (1981). 33 H. Fujii, T. Koyama, and K. Ogura, Biochim. Biophys. Acta 712, 716 (1982).
214
METHODOLOGY
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washed with I0 ml each of chloroform/methanol (2/I), chloroform/acetic acid (3/1), chloroform/methanol (2/1), and chloroform/methanol (2/1) containing 0.1 M ammonium acetate. The last fraction, containing the labeled dolichyl phosphate, is treated with 0.25 volumes water, vortexed, and centrifuged. The lower phase is underpipetted into a fresh tube and taken to dryness for subsequent derivatization or HPLC analysis (see below).
Assays of Dolichyl and Polyprenyl Phosphate Assay by High-Pressure Liquid Chromatography. HPLC of dolichyl phosphate, like dolichol, can be carried out in the adsorption or reversed phase modes. For adsorption chromatography on silica, we use the same column and conditions as described above for dolichol except that the solvent employed is hexane/isopropanol/1.4 M H3PO4 o r H2SO4 (965/35/ 0.5). A typical chromatographic run is shown in Fig. lb. Because of its highly apolar nature, dolichyl phosphate elutes early from this system (6 min) compared to other phospholipids. 34 Quantitation is achieved by preparing a curve generated from injection of standardized solutions (see below). If it is desired to use the HPLC-purified dolichyl phosphate in further studies (e.g., derivatization) in which the phosphoric acid would interfere, the sample can be treated with 1.5 volumes of isopropanol/ water (890/585). The resulting upper phase will contain all the dolichyl phosphate while the phosphoric acid quantitatively extracts into the lower phase. For the reversed-phase HPLC of dolichyl phosphate, the system developed by Chaudhary et al. 3sis employed. For preparation of solvent, 1 g crystalline H3PO4 (Fluka) is dissolved in 1 liter of isopropanol/methanol (1/1). The column and conditions are the same as for reversed-phase analysis of dolichol. As with dolichol, reversed-phase chromatography results in baseline separation of the isoprenolog species (Fig. lc). Assay by Derivatization. This procedure is based on that described by Keller et al.36 and involves the formation of a stable derivative of dolichyl phosphate with [~4C]phenvlchloroformate. O
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R. K. Keller, D. Armstrong, F. C. Crum, and N. Koppang, J. Neurochem. 42, 1040 (1984). 35 N. Chaudhary, D. J. Freeman, J. W. Rip, and K. K. Carroll, Lipids 17, 558 (1982). 36 R. K. Keller, J. W. Tamkun, and W. L. Adair, Biochemistry 20, 5831 (1981).
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DOLICHOL AND DOLICHYL PHOSPHATE
215
(The preparation of the radiolabeled derivatizing reagent, a simple one-step procedure, is described in the original publication.) The purified dolichyl phosphate fraction (usually from DEAE-cellulose chromatography) is taken to dryness in a polypropylene centrifuge tube. To the residue is added 0.1 ml chloroform, 0.01 ml 7 mM triethylamine in chloroform, and 0.01 ml (I0 nmol, about 500,000 dpm) [~4C]phenyl chloroformate in hexane. The sample is taken to dryness at room temperature and dissolved in a small volume (10 ~1) of chloroform/methanol (2/1) and spotted on a silica thin-layer plate. The choice of developing solvent is based on the ambient humidity: with low relative humidity we use chloroform/methanol (7/1) while with high relative humidity acetone is employed. In a separate lane is spotted a standard, prepared from a reaction using unlabeled materials. Radiochromatogram scanning of the sample lane after development reveals a peak at the origin (unreacted dolichyl phosphate), a peak at Rr = 0.5 (the derivative), and two faster moving peaks near the solvent front which are derived from unreacted [14C]phenyl chloroformate. The derivative peak can either be subjected to sample oxidation 36 or scraped and eluted with 1.0 ml hexane/isopropanol/ water (2/6/1.5). From the 3H dpm (derived from the [3H]dolichyl phosphate added to the original sample) and the Jac dpm, the nanomoles of dolichyl phosphate in the sample is determined: (nmol Dol-P/g) =
3H dpm added/g ~4C dpm in derivative × 3H dpm in derivative [r4C]phenyl chloroformate sp. act. (dpm/nmol)
If it is deemed necessary to substract the mass of the [3H]dolichyl phosphate tracer added to the original sample, the procedure is analogous to that described above for dolichol derivatization. The [14C]phenyl chloroformate assay is rapid and allows more samples to be processed per unit time than HPLC analysis, since many derivatives can be applied to TLC plates in a single development. The sensitivity is comparable, since the specific activity of commercially available [14C]phenol (the starting material for the [14C]phenyl chloroformate synthesis) is on the order of 22,000-150,000 dpm/nmol. One possible disadvantage of derivatization is that the partially purified sample may contain contaminants which interfere with the phenyl chloroformate reaction. Such interference would indicate HPLC as the method of choice for quantitation of these samples.