ANALYTICAL BIOCHEMISTRY 66, 330-339 (1975)

Simple Procedures for Determination

of [‘4C]HydroxyproIine

NELLY BLUMENKRANTZAND GUSTAVASBOE-HANSEN

The University of Copenhagen Department of Dermatology (with Connective Tissue

Research Laboratories), Righospital, 2100 Copenhagen, Denmark

Received August 13, 1974; accepted January 14, 1975

Two improved and simplified procedures are presented for determination of [Wlhydroxyprohne. Simplification is achieved by boiling sampies without pre- vious toluene extractions and, after boiling, passing the toluene extracts through a silicic acid column.

Procedure I avoids incomplete toluene extraction of [Wlproline derivatives and uses instead a specific adsorption to a silicic acid column.

Procedure IIA introduces inter alia the silicic acid column to reduce the inter- ference of incompletely extracted [W]proline.

Procedure IIB simplifies procedure IIA by replacing extraction of [W]pro- line derivatives with a silicic acid column.

The new procedures are simple, handy, specific and reproducible methods for determination of [Wlhydroxyproline and are preferable to any other method known today. Procedure IIB is specially recommended for routine use.

The [‘“Cl hydroxyproline (Hyp) assay of Juva and Prockop (l), which is a modification of the Peterkofsky and Prockop (2) and the Prockop er al. (3) methods, is widely used in studies of collagen biosynthesis. How- ever, as the rationale of some of the steps is unclear, an analytical study of the procedure as well as of the method recently published by Rojkind and Gonzalez (4) was undertaken. As a result, considerably simpler methods were developed.

MATERIALS AND METHODS

Sodium pyrophosphate (E. Merck A. G., Darmstadt). A 0.2 M solu- tion in water was adjusted to pH 8.0 with HCl. Solutions adjusted to pH 4.0, 6.0 and 10.0 were also prepared.

Tris bufer. (E. Merck A. G., Darmstadt). A solution of Tris (1.0 M) in distilled water was adjusted to pH 8.0 with HCl. Solutions adjusted to pH 4.0, 6.0 and 10.0 were also prepared.

Sodium acetate-citric acid bufler, pH 6.0. Fifty grams of citric acid, 15 ml of glacial acetic acid, 120 g of sodium acetate and 34 g of sodium

330 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

DETERMINATION OF [14C]~~~~~~~~~~~~~~ 331

hydroxide were dissolved in water and brought to a volume of 1 liter. Toluene was added to avoid bacterial growth, and the buffer was kept at 4°C.

Chloramine T (E. Merck A. G., Darmstadt). Solution I: A 0.2 M solu- tion in water was prepared daily before use. Solution II: A 0.05 M solu- tion was prepared in a mixture of two parts of distilled water, three parts of methyl cellosolve and five parts of sodium acetate-citric acid buffer, pH 6.

Silicic acid. Columns were prepared with silicic acid sized to eliminate small particles. Silicic acid, 1.5 g, was added to 5 ml of toluene, and the slurry was poured in a 10 X 300-mm glass column with a coarse sin- tered-glass disk at the bottom. Columns without sintered-glass disks but plugged with glass wool were also used.

Pyrrole (Fisher Scientific Co.). A 0.1 pmole/ml standard solution in toluene was prepared. Pyrrole solutions of 0.02 and 0.04 pmole/S ml toluene were used as standards for color recoveries.

Zmino acids. L-[14C]proline (Pro) uniformly labeled, more than 180 &i/@mole (New England Nuclear Corp.); [ 14C]Hyp, DL- [ 2-'"C] Hyp, 30-50 mCi/mmole (The Radiochemical Centre, Amersham). Dilutions of both 14C-labeled imino acids were prepared and used for assay, either alone or in mixture.

L-Pro (E. Merck A. G., Darmstadt). A solution containing 1 mg per ml in distilled water. L-Hyp (E. Merck A. G., Darmstadt). A solution containing 2 mg per ml in distilled water.

Ehrlich’s reagent (E. Merck A. G., Darmstadt). The reagent was prepared as indicated by Juva and Prockop (1).

Collagenuse, purified (Worthington Biochemical Corp.). Phosphor solution. Six grams of 2,5 diphenyloxazole (PPO, Packard

Instrument Co.) and 20 mg of 1,4-&s- [2-(4-methyl-5-phenylox- azolyl)] benzene (POPOP, Packard Instrument Co.) were dissolved in 1,000 ml of toluene (E. Merck A. G., Darmstadt) and 600 ml of ethylene glycol monomethyl ether (methyl cellosolve, E. Merck A. G., Darm- stadt) added. Sodium thiosulphate (E. Merck A. G., Darmstadt). Solu- tions of sodium thiosulphate 0.05, 0.1, 0.72, 4, 5, and 7.2 N in water. Potassium thiocyanate (E. Merck A. G., Darmstadt). Aqueous solutions of potassium thiocyanate with a normality of 0.0025, 0.005, 0.0083, 0.0125, 0.0250, 0.05, 0.1, 0.72, 5.0 and 7.2 were prepared. Sodium pyrosulphite (E. Merck A. G., Darmstadt). A 2 N solution in water. Sodium sulphite (E. Merck A. G., Darmstadt). A 2 N aqueous solution. Ammonium sulphate (E. Merck A. G., Darmstadt). A 2 N solution in water. Ferrous sulphate (E. Merck A. G., Darmstadt). A 2 N solution in water. Dithiothreitol (E. Merck A. G., Darmstadt). A 0.05 M solution in water. Thiourea (E. Merck A. G., Darmstadt). A 2 M solution of

332 BLUMENKRANTZ AND ASBOE-HANSEN

thiourea in water. Cysteine HC1 (E. Merck A. G., Darmstadt). Both 0.5 and 0.2 N solutions of cysteine in distilled water.

[ ‘“C]Hyp Assay

Procedure I (Modification of Juva and Prockop’s method (I)). One milliliter of the solution of each carrier imino acid was added to 4 ml of a 14C-labeled hydrolyzed and evaporated sample (1) or a 14C-labeled stan- dard. The volumes were brought to 8 ml with distilled water, 6.0 ml of 0.2 M pyrophosphate buffer was added, and the samples were oxidized with 1.0 ml of chloramine T solution 1. After 20 min, 6.0 ml of 7.2 N

sodium thiosulphate was added. Four milliliters of Tris buffer were added, and the samples were saturated with NaCl or KCI. (Saturation with salt can also be performed before the oxidation step.)

The tubes were immediately sealed and heated in boiling water for 25 min. The samples were cooled, and 12 ml of toluene was added. (Tol- uene can also be added before the boiling step.) The tubes were shaken in a Marius shaker (Utrecht) for 5 min and the samples centrifuged at approximately 600 rpm for 5 min or left a few minutes until the aqueous and organic layers separated. Ten milliliters of the toluene phase were passed through a silicic acid column as indicated by Juva and Prockop (1). The remaining aqueous-toluene phase was shaken for 5 min with 10 ml of toluene, and, after separation of 10 ml of the organic phase, it was also passed through the same column. A third extraction was performed with 5 ml of toluene, and the shaking and passing through the column of 5 ml of the toluene phase was repeated. The final elution volume was 25 ml. Twenty milliliters of the 25-ml toluene elution were counted in a Beckman liquid scintillation counter after addition of 1 ml of phosphor solution. To obtain the recovery, a calorimetric determination of Hyp was made on 0.1 ml of the 25-ml toluene elution. The sample was diluted to 5 ml with toluene, and 2 ml Ehrlich’s reagent was added. After 30 min, the absorbance of the chromogen was read at 560 nm against a reagent blank. The amount of pyrrole originating from the oxidation of Hyp was calculated by using pyrrole standards of 0.02 and 0.04 PM con- centration or by using a standard of Hyp of similar concentration. Taking the percentage color recovery into consideration, a factor was obtained, which was used to multiply the dpm of the [14C]Hyp pyrrole derivative. In case uniformly labeled [14C]Hyp was assayed, it was con- sidered that only four out of five 14C are present in the pyrrole deriva- tive. The fact that 20 ml of the 25-ml elution volume was counted also received consideration. The recoveries calculated by the color reaction and by the use of a [14C]Hyp standard were compared. The same assay can be performed without addition of unlabeled imino acids, only using a [14C]Hyp standard. Calculations were then related to the recovery of the 14C-labeled pyrrole derivative of the [14C]Hyp standard.

DETERMINATION~F [14C]HY~R~XY~R0L~~E 333

The effect of the addition of NaCl or KC1 (1, 5) to saturation after or before the oxidation, as well as the action of the same buffers brought to pH 4,6 and 10 or other buffers (Tris (l), borate (5), borate-alanine (5) alanine (5), sodium citrate (4)) and distilled water instead of pyrophos- phate-Tris buffer were examined. The effect of bringing the samples to alkalinity twice (1) or not bringing to alkalinity at any moment was also tried.

The completeness of [‘“C]Hyp pyrrole derivative recovery was deter- mined by performing one (1) or repeated toluene extractions. To deter- mine its effect on the [14C]Hyp recovery, 3 mg of hydrolyzed serum al- bumin was added instead of unlabeled imino acids.

Alkaline or acid hydrolysis of the collagenase-digested peptides la- beled with [‘“C]Hyp (6) was performed to study the possibility of using the former hydrolytic treatment when glycosylated [‘“C]Hyp is to be determined. Assays were performed on collagenase-digested peptides obtained from lo-day-old chick embryo tibias labeled with [14C J Pro.

Procedure IlA (modi&ation of Rojkind and Gonzalez’s method (4)). Four milliliters of 14C-labeled sample was mixed with 2 ml of chloramine T, solution II. After 20 min the oxidation was terminated by the addition of 1 ml of 4 N sodium thiosulphate or an equal volume of 0.05 N potas- sium thiocyanate, and mixing was performed in a Vortex mixer. The solution was then saturated with NaCl or KCl. Seven milliliters of toluene were added, and the samples were mixed in a Vortex mixer. After the two layers had separated, 5 ml of the toluene phase was trans- ferred to a counting vial. Three additional toluene extractions of 5 ml each were performed. Five milliliters of the toluene phase were taken out of each extract, and the extracts were pooled to a total volume of 20 ml of extract; 1 ml of scintillation (phosphor) solution was added and the fluid counted as [14C]Pro. The rest of the toluene phase was discarded. The aqueous phase containing [14C]Hyp was then converted into the ‘“C-labeled pyrrole derivative by boiling for 30 min in a water bath. After cooling, the samples were extracted with toluene four times, as in- dicated above for [14C]Pro, with the only difference being that the ex- tracts were passed through a silicic acid column as indicated in proce- dure I. To 20 ml of the toluene extracts, 1 ml of scintillation solution was added and the fluid counted in a Beckman liquid scintillation counter. Recoveries were calculated in relation to 14C-labeled Pro and Hyp standards as well as to the total 14C dpm added. They were counted on a 0.1 ml aliquot of each sample analyzed. Recoveries can also be calculated by a color assay as in procedure I, if carrier Hyp is added.

Procedure IIB (modiJcation of procedure [IA). In case [‘4C]Pro recovery was not required, a simplified procedure was performed by omitting the toluene extractions after oxidation with chloramine T at room temperature. The samples were thus oxidized. and, after stopping

334 BLUMENKRANTZ AND ASBOE-HANSEN

the oxidation by addition of 7 ml of sodium thiosulphate and saturation with NaCl or KCl, they were boiled for 30 min. The samples were cooled and extraction of the pyrrole derivative of [14C]Hyp was per- formed as indicated in procedure IIA. The toluene extract was passed through a silicic acid column. To 20 ml of this extract 1 ml of scintilla- tion solution was added and the fluid counted.

E@ect of diRerent substances added after oxidation of [“C]Hyp with chloramine T. Different concentrations of Na2S203, Na2S205, thiourea, Na,SO,, cysteine, KSCN, dithiothreitol, (NH,),SO, and FeS04 were tried as reducing substances. The reaction was performed on a [14C]Hyp standard added to unlabeled Hyp. The recovery of dpm and absorbance was checked.

RESULTS AND COMMENTS

Procedure I

The recoveries of [14C]Hyp calculated by using a [‘“C]Hyp standard or the calorimetric assay for Hyp were similar. This was true of experi- ments performed with different buffers, with pyrophosphate-Tris buffers at pH 4.0, 6.0, 8.2, and 10.0 or distilled water provided salt was added to saturation before or after oxidation and provided the toluene was added before or after the boiling step (Table I). The recoveries were similar provided a recovery factor obtained either by [14C]Hyp or color

TABLE 1 COMPARISON OF RECOVERIES BY THE JUVA AND PROCKOP ASSAY USING

7.2 N NA,S,O, OR 0.0s N KSCN

Added dpm

[ “‘CIHYP [ W]Pro Before boiling

Dpm

Recovered

After boiling

Absorbance Dpm

11,880 385 23,680 2.57 34,000 284 34,000 140,440 31,621

140,440 36,446 KSCN

1,707 395

2,588 25,802 25,442

0.425 4,299 0.422 8,074 0.410 13,820 0.465 13,717 0.420 217

0.377 3,608 0.425 7,578 0.375 10,802 0.370 10,644 0.380 228

DETERMINATION OF [14C]~~~~~~~~~~~~~~ 335

standard (pyrrole or carrier Hyp) was introduced. (In case alanine or Tris buffer alone was used, lower counts and color were obtained, but, after correction according to standards, similar recoveries were ob- tained.) No difference in recovery of [ 14C] Hyp was found in the absence of unlabeled imino acids or in the presence of hydrolyzed albumin, in relation to the usual conditions of the procedure, i.e., in the presence of 2 mg of Hyp and 1 mg of Pro. Experiments performed as indicated by Juva and Prockop, i.e., with one toluene extraction after oxidation at room temperature in the presence of carrier imino acids (1) as well as in their absence, showed that by either procedure, there was incomplete extraction of the [14C]Pro added. The extraction accounted only for 19-28% of the radioactivity. In contradistinction, 54-55% of the radio- activity added was recovered after three toluene extractions and in the absence of carrier. If the silicic acid column was used, only 0.18% of the radioactivity of [14C]Pro was extracted after the boiling step, while 8.5% of the total counts interfered with [14C]Hyp if the column was not used (2). To get complete extraction of oxidation products of [ 14C ]Hyp at least three toluene extractions were required. No difference in recovery of [ 14C] Hyp was found whether chloramine T was dissolved in water, methyl or ethyl cellosolve or in citrate-acetate buffer and methyl cellosolve. Neither was there any difference in recoveries if two- or threefold amounts of chloramine T were used.

A very low recovery of [‘“C]Hyp derivatives was obtained in case sodium thiosulphate was omitted, no chromogen being obtained after the addition of Ehrlich’s reagent. Potassium thiocyanate can substitute for sodium thiosulphate (Table 1).

Alkaline or acid hydrolysis of the collagenase-digested peptides la- beled with [14C]Pro was performed to study the possibility of using the former treatment when glycosylated Hyp is to be determined. Assays were performed with procedure I and with the Juva and Prockop assay on collagenase-digested peptides obtained from 1 O-day-old chick embryo tibias labeled with [l”C]Pro and digested with collagenase (6). No dif- ference in recovery was found whether samples were hydrolyzed with 6 N HCl or 2 N KOH (6).

Procedure IIA

High recovery of proline oxidation products was achieved with this procedure (Table 2). The silicic acid column step minimized the interfer- ence of [ 14C] Pro still unextracted with the four toluene extractions after oxidation at room temperature. Very low recoveries were obtained in case the addition of sodium thiosulphate was omitted. If potassium thiocyanate was used instead, a considerably lower concentration (0.05 N) was required to give similar recoveries as with the regular procedure

336 BLUMENKBANTZ AND ASBOE-HANSEN

TABLE 2 COMPARISON OF RECOVERIES AS [Y]PRo WITH DIFFERENT ASSAYS~

Added dpm Recovered dpm

[ WHYP [ W]Pro Juva and Prockop* Rojkind and Gonzalez’ Procedure IIAd

1.3 x 104 309 37.5 562 2.6 x IO* 615 599 898 3.9 x 104 948 90.5 1.357 3.9 x 104 + 1.5 x 105 28,960 67,898 124,426

1.5 x 105 28,665 68,202 123,260 3.0 x 105 57,262 159.006 245,865 4.5 x 105 88,526 242.644 367.249

(2 r4C extracted before the boiling step. No corrections for recoveries were made. b,c One toluene extraction. d Three toluene extractions pooled, taken to 20 ml with toluene and added to 1 ml of

scintillation solution.

(4 N thiosulphate; Table 3). In addition to thiosulphate and thiocyanate, other substances have been tried to stop the oxidative step. Less effec- tive are sodium sulphite, ferrous sulphate and ammonium sulphate (not shown) as evidenced by recovery of color or [‘“C]Hyp pyrrole deriva- tive. With the procedures of Juva and Prockop or Rojkind and Gonzalez

TABLE 3 EFFECT OF Na,S,O, AND KSCN ON [W[HYP RECOVERY~

Added Hyp Absorbance (dpm = 15,040) at 560 nm

Recovered dpm

0.05 N 0.1 N 0.72 N 4.0 N 5.0 N 7.2 N

KSCN 0.05 N 0.1 N 0.72 N 4.0 N 5.0 N 7.2 N

0.010 986 0.042 1,247 1.830 11,255 1.775 11,393 1.790 11,320 1.830 11,284

1.660 9,151 1.600 9,100 1.570 8.834 1.355 7.456 1.300 7,321 1.290 7,108

a Assay performed as indicated for procedure IIA. Two milligrams of Hyp were added to determine the recovery of the pyrrole derivative with Ehrlich’s reagent. Only one toluene extraction was performed.

DETERMINATION OF [14C]~~~~~~~~~~~~~~ 337

TABLE 4 COMPARISON OF DIFFERENT PROCEDURES FOR [ T]HYP ASSAY u

Added dpm Recovered dpm

[WHYP [ T]Pro

1.3 x 10” 2.6 x 10” 3.9 x 10” 3.9 x 101 1.5 x 105

1.5 x 105 3.0 x 105 4.5 x 105

Juva and

Prockop Proce- dure I

Rojkind and

Gonzalez Proce-

dure IIA Proce-

dure IIB

5622 7320 5113 5255 7937 11,932 14,070 10,055 11,755 14.475 19,425 21,563 17,233 17,348 21,638 22.977 20,716 18,906 18,075 21.440

200 200 1281 71 180 27.5 208 2281 103 200 243 214 5433 132 280

‘I Recovery as [ T]Hyp after boiling. No corrections were made for recoveries.

as well as with our procedure IIA, 2.3-3.5% of the counts of [r4C]Hyp were lost with the toluene extraction before the boiling step.

Procedure IIB

This simplifies our modification of the Rojkind and Gonzalez assay. A strict improvement was achieved by introduction of the silicic acid col- umn in procedure HA. The boiling step without previous extraction of the derivatives of [14C]Pro makes the procedure quicker than IIA. The method is simpler than any other method for [‘“C]Hyp (Table 4).

In procedures IIA and IIB a high recovery of counts originating from [‘“C]Hyp and [‘“Cl Pro, which were not retained by the silicic acid col- umn, was obtained in case the oxidation was ended with four extractions with toluene at room temperature without addition of Na2S20s. Op- timum recoveries of the pyrrole derivative of [‘“C]Hyp and unlabeled Hyp were obtained by using 1 M Na,S,O, or 0.05-0.1 M KSCN. Lower recoveries were obtained with the other substances examined. In case carrier Hyp was added, no chromogen was obtained upon addition of Ehrlich’s reagent to an aliquot of the toluene extract when Na&O, or KSCN were omitted.

There was no difference in recoveries whether columns plugged with glass wool or with sintered-glass disks were used.

DISCUSSION

With the assay of Juva and Prockop, indicating one toluene extraction, 19-28% of the radioactivity of [‘“Cl Pro was recovered. By using the Rojkind and Gonzalez procedure, also indicating only one toluene ex- traction, a 54-55% recovery of the oxidation products of [‘“Cl Pro was

338 BLUMENKBANTZ AND ASBOE-HANSEN

obtained. Two further extractions were found necessary to remove the proline oxidation products, but they were not counted (4). Peterkofsky and Prockop (2) and Switzer and Summer (7) also used only one toluene extraction for the measurement of the radioactivity of [14C]Pro. How- ever, to remove the remaining radioactivity of the proline oxidation- products, the former authors used three or four additional extractions which were discarded. Our results, after using only one toluene extrac- tion before the boiling step, were in agreement with those of the above- mentioned authors (2, 4). The need of an exhaustive extraction for measurement of the radioactivity of [14C]Pro (procedure IIA) made us perform three or four toluene extractions. The extracts were pooled and counted. The yield turned out to be highly increased, bringing the recov- eries to 80%. As indicated by Peterkofsky and Prockop (2) it appears that one carbon of [‘“Cl Pro is lost as [ 14C] 0, during the oxidation and that the radioactivity counted presumably originates from Al- [14C]pyrroline. On this basis, our recovery of 80% would account for a 100% yield, if we take into consideration that we are counting the radioactivity of four of the five original carbons of [14C]Pro.

A comparison of the radioactivity recovered from the [14C]pyrrole derivative of Hyp by performing our simplified assays and those of Juva and Prockop and Rojkind and Gonzalez showed higher recoveries with our procedures. This might be due to the fact that some radioactivity of [14C]Hyp is lost when extracted together with the [‘“C]Pro in the toluene extraction prior to the heating step required in the two latter procedures and procedure IIA (Table 2). In the two simplified proce- dures for [14C]Hyp (I and IIB) these extractions are omitted, the silicic acid column being the only necessary step to eliminate, by adsorption, the interference of [‘“C]Pro in the final counts. The interference of [14C]Pro in the final counts of the pyrrole derivative of [14C]Hyp is higher in the Rojkind and Gonzalez procedure than in the Juva and Prockop or in our modifications. The former assay is performed without passing the toluene extract through a silicic acid column. The introduc- tion of this step in the assay of Rojkind and Gonzalez reduced the inter- ference of [‘“Cl Pro.

In a previous study we noticed the need of repeated toluene extrac- tions for quantitation of Hyp by a calorimetric assay (8). The rationale of two different salts used by Juva and Prockop and by Kivirikko et al. (5) to saturate the samples, i.e., NaCl for the [14C]Hyp assay (1) and KC1 for the calorimetric method (5), both based on the same procedure, is not clear. NaCl or KC1 was used randomly by us on different aliquots of the same sample submitted to the [‘“C]Hyp or the calorimetric assay. There was no difference in the recovery in parallel determinations. It is necessary to stress the importance of the loss of 2.3-3.5% of the counts

DETERMINATION OF [ 14c] HYDROXYPROLINE 339

of [14C]Hyp which are extracted as [‘“Cl Pro when extractions are per- formed prior to the boiling step. It seems possible that, in addition to reducing chloramine T (l-5), thiosulphate and thiocyanate may act by some other mechanism. The difference in the optimum amounts required for maximum yield as color or [‘“Cl Hyp derivative extracted and the fact that, in the presence of sodium sulphite, lower recoveries were ob- tained, suggest that, besides reduction, a different mechanism is in- volved. As acid and alkaline hydrolysis of aliquots of the same sample of “C-labeled collagen synthesized by chick embryos have shown similar [‘“C]Hyp counts, no glycosylation of this Hyp like that known from plant glycoproteins (9-l 1) can be assumed to take place.

The use of a Hyp standard instead of, or in addition to, pyrrole for calculation of recoveries based on calorimetric assay is advantageous because it must pass all steps of the procedure with the same reagents except, if so wanted, the passing through the silicic acid column. The pyrrole standard gives a practically constant chromogen with Ehrlich’s reagent, only dependent on the reagent. As similar recoveries are ob- tained without the use of unlabeled Hyp, a standard of [14C]Hyp can be used for calculations with the same advantages as in the calorimetric assay.

ACKNOWLEDGMENT

The expert technical assistance of Mrs. Hanne Bojden is highly appreciated.

REFERENCES 1. Juva, K., and Prockop, D. J. (1966) Anal. Eiochern. 15, 77. 2. Peterkofsky, B., and Prockop, D. J. (1962) Anal. Eiochem. 4, 400. 3. Prockop, D. J., Udenfriend, S., and Lindstedt, S. (1961) J. Biol. Chem. 236, 1395. 4. Rojkind, M., and Gonzalez, E. (1974) Anal. Biochem. 57, I. 5. Kivirikko, K. I., Laitinen, O., and Prockop, D. J. (1967) Anal. Biochem. 19, 249. 6. Blumenkrantz, N., and Prockop, D. J. (1969) Anal. B&hem. 30, 377. 7. Switzer, B. R., and Summer, G. K. (1971) Anal. Eiochem. 39, 487 (1971). 8. Blumenkrantz, N., and Asboe-Hansen, G. Anal. Biochem. 63, 331 (1975). 9. Lamport, D. T. A. (1967) Nature (London) 216, 1322.

10. Chrispeels, M. J. (1970) Biochem. Biophys. Res. Commun. 39, 732. 11. Allen, A. K., and Neuberger, A. (1973) Biochem. J. 135, 307.