Modified procedure for the assay of H3- or C14-labeled hydroxyproline

ASALYTIC4L

BIOCHEMISTRY

15, 77-83

(1966)

Modified Procedure H::- or Cl’-Labeled

for

the

Assay

of

Hydroxyproline

13ccauac l~ydroxyl~rolinc can bc oxidized to pyrrolc in :L rclativcly specific reaction (l-31, it has been possible to devise siliiplc :wwy methods for radioactivclly 1n1~M h~drosg’proline which do not rcquirc preliminary chromatogwplric isolation of the imino acid (4, 5). Owl t,hc past several years we have modified the two procedures previously published (4, 5) SO that the assay is simpler, ~uorc reproducible, and less :LffcTtctI by t11c pl‘c’scncc of 0th c’l’or H,‘-lnf)clctl compounds. Althougll the preeent~ proccclurc nlexwres tot,nl rndioacti\-e liydrosyl~rolinc rather than the specific rarlioactirity of hyclrospl~rolinc (4). it is mow readil::ipplic:kble to samples from a variety of sources. METHODS

Materials. The following r solutions were used: 0.2 X chloramine T ( I~ktninn Organic Chcniicals) prepared in distilled water just Iwforc USC; 0.2 ;I1 sodium pyroJ~hoephate (Fisher Certified reagent) adjusted to pII 8.0 with HCl; 3.6 31 Fodium thiosulfatc (Fisher Certified rcngent) clissolwd in distilled water and stored under tolucne at room tempernturc for up to 1 month; l.ON tris bufYcr (Fischer Primary St)andnrd) in clistilled water adjusted to pH 8.0 with HCI; silicic acid (Bio-Rat1 Laborntories) ; tohicne (Americ:m Clicniicnl Society spccificat~ions). p-I~ii~~ct~li~laminol,enznItlcli~dc or Ehrlich’s rexgent (practical gratlc, Eastman Organic (IJiciuic:~ls i was rwry~&~llizcd ikicc from hot :il)solute ethanol, and the solution was prepared as described preriously (G) . The phosphor solution for liquid scintillation counting wae also prepared as clcscribed previously (4). Pyrrole (practical grade, Fisher Scientific Co.) was purified by fractional distillation. Stantlar(1 solutions of 0.1 /mole/ml were preljared by ‘Present, address: Turku, Finland.

Department

of

Medical 77

Biochemistry,

liniversity

of

Turku,

dilution of freshly distilled lneln~rations wit11 tolurne. and they were stored at --20” for several months. Assa,f/ Procedwe. Samples for assay were hydrolyzed by autoclaving at 120” overnight in enough 6N HCl to provide a final protein concentration of less than 10 mg/ml. If significant amounts of humin formed during the hydrolysis, a “humin precipit,ant” (6) was employed. The hydrolyzatcs wre evnl~oratcd to dryness in z’acw, and the residue was dissolved in 4.0 ml distilled water. A 0.2- to 0.4-ml aliquot was taken for assay of total C” or H’: content (7). An aliquot containing leas than 80,000 disintegrations per minute idpm) of C” or lcus than 150,000 dpm of H” was selected for aas:ly of radioactive hydroxyproline (title infix). The nliquot was 1~lnccd in a screw-capped test tube (Kimblc 45066-A, 200 X 25 mni 0.~1.1, and 1.0 mg L-proline and 2.0 mg I,-hyclroxy1~roline (General Biochtwicale) wre added from concentrated stock solut’ions. The pH was adjusted to 8.0 with sodium hydroxide solutions, and the volume was maclc: up to 8 ml with distilled water; 6 ml of 0.2 M sodium pyrophosphatc huffcr, pH 8.0, was added, and the sample was oxidized by adding 1.0 1111of freshly prepared 0.2M chloraminc T solution. After oxidation at room t~cmperaturc for 20 min, the reaction ~vas terminated by adding 6.0 ml of 3.6~11 sodium thiosulfate solution. One drop of 1% phenolphtl~alcin in ethanol was added, and the pH of the solution was adjusted t,o a faint pink color with 1.0 and O.lLV sodium hydroxitle. Then 4 ml of I .OM tris l~uffer, pII 8.0, was added, and tllc sample was saturated with cxccss SaCI. Tolucne (10 ml) was added, the tube was scaled with a Teflon-lined plastic cap, and the sample was shaken vigorously for at least 5 min. The sample was centrifuged briefly at about 600 X g, and the toluene layer XIS completely removed with suction through a capillary pipet. The tube was again sealed, and then heated in a briskly boiling hot water bath for 25 min. The sample was cooled to room temperature, and, if necessary, additional NaCl adclcd to keep the sample saturated. Exactly 12.0 ml of toluene was added, and the sample again shaken vigorously for at least, 5 min. The sample was centrifuged briefly at about 600 X g, and 10.0 ml of the tolucne layer WEW placed on a short column of silicic acid. The column was prepared by pouring a slurry of 1.5 gm of silicic acid in 5 ml of toluene into a 10 X 300 mm glass column with a coarse sintered-glass disk. In order to prevent clogging, a thin layer of sand was placed on the disk. After the sample passed through the silicic acid, the column was eluted with 5.0 ml and then with 10.0 ml of toluene. The tot,al effluent from the column was pooled, and it was made up to 25.0 ml with toluene. A 20.0-ml aliquot of the solution was placed in a counting vial with 1 ml of phosphor for assay of radioactivity in a liquid scintillation counter. For the calorimetric assay, a O.l-ml

PROCEDURE

FOR

HTDROSYPROLIh-E

79

aliquot was diluted with 5.0 ml of t.oluene, and 2.0 ml of the Ehrlich reagent was rapidly mixed in with a Vortex mixer. The color was developed at room temperature in the dark for 30 min, and the absorbance at 560 ml,, was measured against a reagent blank. The amount of pyrrole in the sample was calculated by reference to the absorbance of pyrrole standards of 0.02 and 0.04 ,mole which were dcvelopcd at, the same time. ?L control which contnincd 1 mg of l)rolinc, 2 mg of hydroxyproline, or 1.50~000 clpm proline-3,4-H3 (NW and 80,000 tlpm L-proline-C” England Xuclwr Corp.) was assayed with cnch series of samples ivide infw ) . ~‘nlotlntiorzs. The following formula was used to calculate the amount in tile aliquot~ oxidized from the observed cpm in of hydroxyproline-C’” final solution of pyrrolc-Cl’: observed cpm X !gF X $ X : X T = dpm Hypro-Cl3 1 where E is the efficiency of the counting system, 25/20 is the correction for aliquot of effluent n-hieh was counted, 5,/4 is the correction for the loss of the carboxyl carbon in the conversion of uniformly labeled liyclrosyproline-Cl’ to pyrrole-Cl’, and IZ is the per rent recovery of hydroxyproline as pyrrolc in the assay. Total dpm IlvclrosSproline-C1‘~ in the sample was obtainwl by correcting for the aliquot which was taken for oxidation. When liy~1~~os~proline-3,4-I-I~~ was assayed, the correction factor for t’he loss of isotopic label in the conversion to pyrrole was 3/2 instead of 5/4 (81.” The per cent recovery of llydroxyproline as pyrrole (R) was calculated from the colorimct,rir away with the formula : b)(~I&, m X wolr,, x 6 x pmolb Hyp ’ loo = ’ PS where OD,, is absorbance of pyrrolc from t,hc sample, OD,, is absorbance of a standard solution of pyrrole, /mole,,, is the amount in ;Lmoles of pyrrole standard (usually 0.04 ;mole), 25/0.1 is the correction for the aliquot of toluene effluent taken for calorimetric assay, and ,mole Hyp is the amount in ,kmoles of carrier hydroxyproline uwd in the assay (15.2 ,umoles) RESULTS

AYD

DISCUSSION

Adequate recoveries in various oxidative assay procedures for hydroxyproline depend on an optimal relationship between the amount of oxidant ‘In situations in which the total incorporation of L-proline-3,4-H’ into protein was compared to the incorporation of L-proline-3,4-H’ into hydroxyproline-H3, a correction factor of 4/2 has bwn used (9), since the H” on one hydrogen position is lost in the conversion of L-proline-3,4-H’ to hydroxyproline-H’ (8).

80

JCT’B

.IND

PROCKOP

and the amounts of oxidizable materials present in the sample (6). Insufficient amount:: of oxidant result in incomplete oxidation of the hydroxyproline, but excessive amounts also result in low yields because, of overoxidation. The procedure originally published for assay of labeled hydroxyproline (4) provided a met.hod for obtaining directly the specific activity of the imino acid in biological materials. In practice, however, a number of assays wit#h varying amounts of chloramine T had to be performed on aliquots of the same sample in order to establish optimal conditions. The present procedure is limited to measuring the total content of H3- or CWhpdroxyproline in samples, but the modifications introduced make it possible to employ standard conditions in assaying many different types of samples. As shown in Table 1, values obtained were constant with a threefold variation in the amount of oxidant which was employed. The per cent

EFFECT OF VARYISC AMOTJSTS OF OXIDANT AYD EMBRYONIC HOXOCENATE OS THE Bssau PROCEDURE Chlornmine for oxidation. n1m01es

H?O

Hydrolyzate: 6.0 mlc 3.0 ml 1.5 ml 0.8 ml

10,500 10,500 10,500 10,500 3 ,800 3 , 800 3 ( so0 3,son

T

Itecovcry,~ %

0.2 0 .3 0 .4 0.6

61.3 58.6 52.7 47.4

n.2 0.2 0.2 0.2

‘)‘> d-.1‘, 30. .5 42.5 49.9

Assnyed Hvp-C’s content. dpm 10 ,600

10,400 10,100 10,200 3,690 3,420 3,870 3,590

a Assays were performed as described in text except that the amounts of chloramine T were varied as indicated. The hydroxyproline-Cn, 9.9 X 10” dpm per rmole, m-as prepared by administering L-proline-C i4 to chick embryos and isolating the hydroxyproline-Cl4 with three chromatographic systems (10). b The per cent recovery of hydroxyproline was calculated as described in the text. This calculation is simplified slight,ly in order to facilitate the computation of dpm and it does not correct for the fact that only 10 ml of the 13 ml hydrosyproline-C14, toluene in the second extra&on is placed on the silicic acid column. If corrected for the pyrrole discarded in this step, the actual per cent recoveries in the assay are 20% higher than the values quoted here. Although chloramine T is a relatively stable reagent, it is occasionally necessary to use more than 0.2 mmoles of some batches to obtain optimal recoveries. c The “hydrolyzate” consisted of a homogenate of lo-day old chick embryos which was centrifuged at 100,000 X g for 1 hr. The supernate was dialyzed, hydrolyzed with 6 N HCI, evaporated to dryness, and dissolved in water. Ninhydrin assays indicated that the final solution comained about 100 mg amino acids per ml.

PROCEDURE

FOR

81

HTDROSTPROLINE

recovery in the procedure tlecrcawd when samples contained more than about, 50 mg amino acids, but the assayed value for labeled hydroxyproline was not greatly affected when samples contained up to 600 mg amino acids and the recoveries mere reduced to less than half the recoveries obtained with pure solutions.” With samples n-hich contained less than 50 mg amino acids, the cnlculnted recoveries rang& from 45 to 65% in over 1000 assays performed over :I period of 1 ycnr. The variation is probably attributable to fluctuat8ions in room temperature and to slight differences in t,he techniques employed by different workers. Within groups of similar samples assayed on the same day, the calculated rccoveries varied by less than t37u. The use of a silicic acid column simplifies the assay procctlurc, because the multiple extractions n-ith tolucnc can be eliminated. The use of the column has also increased the reproducibility of t’he assay. probably because it has eliminated mechanical errors, pH changes, and oththr variations encountered with the longer procedure. Of even greater importance is the finding that use of the column has reduced t’he possible error which might be introduced hy the prcwnce of radioactive conlpoun11:: other than II”- or C14-pyrrolc in the final tolucnc extract (Table 21. TABLE INTERFERENCE

FROM

OTHER

3 L.tRELED

COMPOUNDSa

57” of initial r:tdioactivits

Proline-C1a, Proline-3,4-H3, Hydrolyzate

7.9 X 1W dpm I .3 X lo5 dpm of algae-V. 4.7 X lo6 dpm

31” 30~ 14,400


‘I Bssays were performed as described in test. Hydrolgzat,e of algae-V was obtained from New England Nuclear Corp.; the preparation contained 6% proline-W, and 70% of the total Cl4 content was nccount)ed for by amino acids. Observed cpm are not corrected for the background of the count,ing system, which TKIS 20 cpm for Cl4 and 24 cpm for H3. h Mean of 50 assays with a range of 25 to 37 cpm. c hIcan of 30 assays with n range of 26 to 43 cpm.

variety of samples, several prrcaut8ionL * rhoultl IJe observed. For example, samples must contain less than about 0.5 pmole of hydroxyproiine, be’ .Although the amount with highly solutions.

the possibility was not explored of chlornmine T used in the assay irnpurc~ samples even though this

here, it seems probable that would improve the recoveries would reduce the reroveries

increasing observed with pure

82

JUV$

AND

PROCKOP

cause larger amounts would falsely increase the apparent recovery of pyrrole from t,he 15.2 pmoles hydroxyproline used as carrier. Although the values obtained with highly impure samples are apparently valid (Table I), we have generally verified the results of assays in which the recoveries are less than 40% by partially purifying selected samples and then repeat’ing the asr;ays. In the rase of stud& on the hydroxyproline in collagen, most Pamples can be adequately purified for this purpose by extracting t,he collagen as gelatin with hot ~~atcr or hot trichloroacetic acid. An additional possible source of error is the presence of labeled compounds other than pyrrole in the final toluene extract.. Labeled proline is the most commonly used precursor of labeled hydroxyproline, and the present procedure reduces the interference from radioactive proline to less than 0.1% (Table 2). In order to minimize possible interference from labeled proline, we have generally selected alicluots of samples for assay which contain less than 80,000 dpm C” or less than 150,000 dpm H3 so that, if all the label in the sample is in proline, the observed cpm in the final t,oluene extract will not be significantly greater than the background of the counting system. Also, we have added labeled prolinc to the control that is processed with each series of assays in o&l to establish that contamination from labeled proline has not been encountered. Additional precautions should be considered whrn samples for assal may contain labeled compounds other than proline or hydroxyprolinr. Glutamate-C? was previously shown (4) not to present any difficulty, but assay of a hydrolyzate of labeled algae resulted in a higher level of observed cpm than could be accounted for by the imino acid content of the hydrolyzate (Table 2). Even greater interference might be encountered in more complex samples, but this possible source of error is of little concern if preliminary examination of eclccted samples by paper chromatography indicates t’hat most of the radioactivity is limited to proline, hydroxyproline, glutamate, and other common amino acids. 0111. experience to date suggests that the procedure can be applied directly to most biological materials in which labeled hydroxyproline accounk for at least 2% of the total radioactivity.

Previously published procedures for the assay of radioactively labeled hydroxyproline have been modified so that the assay is simpler, more reproducible, and less affected by the presence of other Cl”- and H3labeled compounds. Although the prcl-cnt procedure measures total

radioactive hydroxyproline rather than the specific radioactivity of hydroxyproline, it is more readily applicable t.o samples from a variet,y of sources .~Cl~iSO\~L~D,C;~lI~s’rs Thr authors gratefully scknowledge the c~spert assistance of Mrs. Tracy Mulveny and ?vIiFs Elizabeth Borton. The work KU snpportcd in part by grants numba 6-Tl-A&15459, HD-00183, rind FR-00107 from the IT. S. I’nl~lic Health ?krrice. REFERBSCES 1. L.ASC, Ix., z. mysiol. 3.

NUJMAN,

4. PROCKOP,

Chem. 219, 148 (1933).

AXD AKABORI, S., 2. PIlysiob. Chcm. 224, lS7 (1934). R. E., ASD Local-, hl. A., J. Viol. Chcm. 186, 549 (1950). D. J., UDEITFRIKXD, d., .ISD LIKDSTIDT, S., J. Viol. C’ilcta. 236, 1395

2. ~~.~LDSCHMIDT-LEITZ,

E.,

(1961). 5. 6.

B., AND PROC~KOI~, D. J., Awl. zZioci~(~m. 4, 400 (1962). J., ASD UDESFIUBND, S., Awl. Bioci~cv~. 1, 228 (1960). 7. PROCKOP, D. J., AXD EBERT. P. P., Awl. Rio&em. 6, 263 (1963). 8. PROCKOP, D. J., EBERT, I’. 8.. ASD SII.WI~O, B. 1\1., Arch. Biochem. ni0pily.s. 106, 112 (1964). 9. PROCKOP, II. J., AKD Jum. K., Hiocllcm. Uiophys. lies. Comnam 18, 54 (1965). 10. I<.~Tz, IX., PIKJCKOP, D. J., .AND~~~~~~~~~~~~~ S., J. Bjol. Chcm. 237, 1585 (1962). PETERKOFSKY, PROCKOP, D.