The absence of iron and certain coenzymes in highly purified protocollagen proline hydroxylase

504

BIOCHIMICAET BIOPHYSICAACTA

BBA 35769 T H E ABSENCE OF IRON AND C E R T A I N COENZYMES IN H I G H L Y P U R I F I E D PROTOCOLLAGEN P R O L I N E H Y D R O X Y L A S E

M A I J A P - ~ N K A L A I N E N AXD K A R l 1. K I V I R I K K ( _ )

Children's Hospital, University of Helsinki, Helsinki ( lrinland) (Received A u g u s t i 3 t h , 197 o)

SUMMARY

The possible presence of protein-bound iron and of certain coenzymes in highly purified protocollagen proline hydroxylase was studied. All three enzyme preparations subjected to the study contained far less than I mole of iron/mole of enzyme. The results indicate that protocollagen proline hydroxylase does not contain stoichiometric amounts of iron bound firmly. The absorption spectrmn of the enzyme showed no absorption maxima between 28o and 6oo m/~, suggesting the absence of certain prosthetic groups, such as flavin nucleotides. Inhibition studies suggested that pyridoxal phosphate is not involved in the enzyme reaction, and direct assays for thiamine indicated the absence of thiamine and thiamine pyrophosphate. Thus, the decarboxylation of a-ketoglutarate coupled to the hydroxylation of peptidyl proline residues does not seem to be catalyzed by these prosthetic groups.

INTRODUCTION

The enzyme protocollagen proline hydroxylase catalyzes tile synthesis of hydroxyproline in collagen by the hydroxylation of proline which has been incorporated into protocollagen, a large proline-rich and lysine-rich polypeptide precursor of collagen (for review, see ref. I). The presence of the activity of this enzyme has been demonstrated in connective tissue from a number of sources, and recently the enzyme has been isolated in a highly purified form from chick embryo extracts 2,3. Purified preparations of the enzyme have an absolute requirement for molecular oxygen, ferrous iron, a-ketoglutarate and a reducing agent which can be ascorbate, other enediols, or tetrahydropteridines (see refs. I, 4, 5). It has been suggested that the ferrous iron is bound loosely to the enzyme ",7, and that metal chelators which block the activity of the enzyme remove the iron from the enzyme. On the other band, some more recent experiments seem to indicate that the iron is very firmly bound to the enzyme, and it can not be removed even after denaturation and subsequent treatment with strong chelating agents 8,9. Biochim. Biophys. Acta, 22q (I97~) 504 5o's

PROTOCOLLAGEN PROLINE HYDROXYLASE

505

The availability of highly purified preparations of protocollagen proline hydroxylase made it possible for us to study the presence of iron in the enzyme by carrying out direct assays for this metal. In addition, an attempt was made to gain some insight into the reaction mechanism by studying the possible presence of certain other prosthetic groups in the enzyme.

METHODS

Protocollagen proline hydroxylase was isolated from chick embryo extracts by procedures reported previously~,Z,1°. All three enzyme preparations used in the study were homogeneous in the analytical ultracentrifuge, and over 90-95 °/o pure in disc electrophoresis1°. Isoelectric focusing showed the presence of only one protein corresponding with the enzyme activity 1°. These enzyme preparations synthesized about 500-700/~g hydroxyproline/mg protein per h with a saturating concentration of the synthetic polytripeptide (L-Pro-Gly-L-Pro)n, molecular weight of 6600, (Miles-Yeda, Kiryat Weizmann, Rehovoth) as substrate, under the standard incubation conditions described previously (see ref. 3). The protein content of the enzyme preparations was assayed by peptide absorption at 225 m/~, and by ninhydrin assays of acid hydrolysates. The iron content of the enzyme preparations was determined by using atomic absorption spectroscopy. Three different atomic absorption spectrophotometers were used (see Table I), and before the experiments, the instruments were carefully adjusted to give optimal results for iron determinations with our standards and bovine serum albumin solutions. The enzyme preparations were dialyzed before the experiments against 0.05 M NaC1, i mM Tris-HC1 buffer (pH 7.8) prepared in deionized, TABLE I IRON CONTENT OF PROTOCOLLAGEN PROLINE HYDROXYLASE

A t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r s used : E x p t . I, P e r k i n - E l m e r Model 3o3 ; E x p t . 2, P e r k i n E l m e r Model 4o5; E x p t . 3, T e c h t r o n Model AA- 5. BSA, b o v i n e s e r u m a l b u m i n .

Expt. No.

Sample

Protein (mg/ml)

Iron found (l~g/ml)

Moles of iron]mole of enzyme

i

Enzyme I BSA

o.15 o.15

2

Enzyme 2 BSA BSA + 0.2/~g/ml Fez+ BSA + 0.4/~g/ml Fe ~+

I.OO I.OO i.oo I.OO

o.28 0.5o

Enzyme 3 BSA BSA + o . I / ~ g / m l Fe z+ BSA + o. 4 / ~ g / m l Fe 2+

1.7 ° 1.5 ° 1.5o 1.5o

o.o 5 o.o9 o.19 0.5o

3

<0.o2

o.o 7

o-o. i *

" I t m a y be n o t e d t h a t t h e iron c o n t e n t of t h e e n z y m e w a s n o t h i g h e r t h a n t h a t of b o v i n e s e r u m a l b u m i n . W e do n o t know, w h e t h e r t h e a b s o r p t i o n o b s e r v e d w a s due t o iron or s o m e artefact.

Biochim. Biophys..Acta, 229 (1971) 5o4-5o8

506

M. PANKALAINEN, K. I. E1VII,HKt~.()

quartz-distilled water. The same buffer was used as the blank solution, and for preparing the standards and the bovine serum albumin solutions. The absorption spectra of the enzyme preparations were determined in a Perkiu Elmer Model I24 double beam spectrophotometer equipped with an automatic recorder. The inhibition experiments with canaline were carried out bv adding various concentrations of this compound to the standard incubation system (sec ref. 3), and by assaying the amount of hydroxyproline synthesized. The thiamine and thiamine pyrophosphate contents of the enzwne preparations welt} assayed microbiok)gically by using Lactobacillus fcrmcnti fly', and chemically by using chr,,matographic assay techniques ~a. RESULTS AND DISCUSSION

Iron content of protocollagen proline hydrox~,lase Studies on the presence of iron in protocollagen proline hydroxylase indicated that two out of three enzyme preparations used in the experiments affected the atomic absorption spectrophotometers slightly (Table I). However, it should be noted that the instruments were used with maximal or almost maximal sensitivity, as the amounts of the enzyme available for the analyses were relatively small. Under these conditions, a similar or even slightly higher absorption was observed with similar concentrations of crystalline bovine serum albumin. It seems possible that at least part of the absorption was due to an artefact caused by the protein content of the samples rather than by the iron, On the basis of the molecular weights of about 56 for iron and about 250 ooo for the enzyme (ref. IO), it can be calculated that even if all the absorption observed was due to the iron, the content of this inetal in all three enzyme preparations was far less than I mole/mole enzyme (Table I). The value was very low, especially in Preparation 3. If part of the absorption was due to an artefact, the correct values are'even lower. The present data indicate that protocollagen proline hydroxylase does not contain iron in such firmly bound form that the enzyme would retain significant amounts of iron during the purification process. The purity of the enzyme prepa-

1600

1200

~ 080C 040C!

°2;

300

400 WAV LENGIH(mp)

500

600

Fig. 1. Absorption s p e c t r u m of protocollagen prolinc hydroxylase, i.5 mg/ml, in a solution containing o.2 M Na('l, o.z M glycine, and o.oi M Tris -HC1 buffer (pH 7.8).

I3iochim. Biophys. ,4cta, 2-'9 (I97 l) 5o4-5 o~;

PROTOCOLLAGENPROLINE HYDROXYLASE

507

rations used in these experiments was quite high (see METHODS),and it does not seem possible that the absence of stoichiometric amounts of iron would be due to the presence or large amounts of iron-flee contaminating proteins. On the other hand, the preparations of protocollagen proline hydroxylase which were studied with radioactive iron and reported to contain very firmly bound ironS, 9 had a much lower degree of purity. It seems possible, therefore, that in those experiments the iron was present in proteins other than the enzyme. Studies on possible coenzymes

Initial attempts to study the presence of prosthetic groups were made by determining the absorption spectrum of the enzyme. The enzyme solutions were completely colourless even in a concentration of 15 mg/ml, and the absorption spectrum in a solution containing 0.2 M NaC1, 0.2 M glycine, and o.oi M Tris-HC1 buffer (pH 7.8) showed no absorption maxima between 280 and 600 m#. Fig. I shows the absorption spectrum of an enzyme preparation containing 1.5 mg protein/ml, and similar results were obtained with the other two enzyme preparations studied in protein concentrations up to 5 mg/ml. These results suggest that the presence of certain coenzymes having characteristic absorption maxima can be excluded. These coenzymes include flavin nucleotides, which are present in several monooxygenases (see ref. I4), and pyridoxal phosphate, which is involved in several decarboxylation reactions (see below). Recent studies on the role of a-ketoglutarate in the protocollagen proline hydroxylase reaction have indicated that a stoichiometric decarboxylation of a-ketoglutarate is coupled to the hydroxylation of peptidyl proline residues 15. This prompted us to study the presence of coenzymes involved in other decarboxylation reactions. Pyridoxal phosphate is involved in several decarboxylation reactions of amino acids in. Recent studies on six pyridoxal phosphate-dependent enzyme reactions have shown that canaline (I-amino-3-amino-oxybutyric acid) strongly inhibits all these reactions17, is. However, we found no inhibition of protocollagen proline hydroxylase with I mM canaline. On the basis of this lack of inhibition with canaline and the absence of characteristic absorption maxima in the spectrum of the enzyme (see above), it seems that pyridoxal phosphate is not involved in the reaction. Thiamine pyrophosphate serves as coenzyme in several decarboxylation reactions of a-keto acids, such as in the decarboxylation reaction of a-ketoglutarate catalyzed by a-ketoglutarate dehydrogenase 16. To study whether this coenzyme is present in protocollagen proline hydroxylase, preparations of the enzyme were assayed for thiamine both microbiologically and by chromatographic procedures. Under the conditions used, the former assays would have detected the presence of 0.05 mole of thiamine or thiamine pyrophosphate/mole of enzyme, and the latter assays of 0.2 moles of thiamine derivates/mole of enzyme. Both assay methods gave negative results with three separate enzyme preparations. Thus, our results indicate that thiamine derivates are not involved as coenzymes in the decarboxylation of a-ketoglutarate by protocollagen proline hydroxylase. ADDENDUM (Received December I4th , 197o ) After the submission of our manuscript, RHOADSAND U D E N F R I E N D 1~ reported Biochim. Biophys. Acta, 229 (1971) 504-508

508

M. PANKAI, AINEN, K. I. KIVIRIKKO

that protocollagen proline hydroxylase purified from newborn rat skin is similar to the enzyme studied here in that it does not exhibit a visible spectrum and does not contain thiamine pyrophosphate. ACKNOWLEDGEMENTS

We are grateful to Mr. T. Putkonen, Phil. M., K a u k o m a r k k i n a t Oy, and Mr. O. Aulio, Phil. Lie., Perkin-Elmer Oy, for assistance in the assays with atomic absorption spectrophotometers, and to Miss Eeva-Liisa Rahiala, Phil. M., for helpful suggestions and for the gift of canaline. This work was supported by grants from the National Research Council for Medical Sciences, Finland, and from the Sigrid Jus61ius t:oundation. REFERENCES i D. J. PROCKOP, in E. A. BALASZ, Chemistry and Molecular Biology of the Intercellular Matrix, Vol. I. Academic Press, L o n d o n and New York, 197o, p. 335. 2 K. I. KIVIRIKKO, J. ]-IALME AND K. SIMONS, in E. A. BALAZS, Chemistry and 3Iolecular Biology of the Intercellular Matrix, Vol. i, Academic Press, L o n d o n and New York, 197 o, p. 411. 3 J- HALME, K. I. KIVIRIKKO AND K. SIMONS, Biochim. Biophys. Acta, 198 (197 o) 460. 4 J- J. HummON, A. L. TAPPEL AND S. UDENFRIEND, Arch. Biochem. Biophys., i18 (1967) 231. 5 K. I. KIVlRIKKO AND D. J. PROCKOP, Arch. Biochem. Biophys., 118 (1967) 611. 6 K. l. KIVIRIKKO AND D. J. PROCKOP, -Proc. Natl. Acad. Sci. U.S., 57 (I967) 782. 7 K. I. KIVlRIKKO, H. J. BRIGHT AND D. J. PROCKOP, Biochim. Biophys. Acta, I5r (1968) 558. 8 J. I:{ENCOVA, J. HURYCH, J. t{OSMUS AND M. CHVAPIL, Abstr. 5th ~leeting, Federation ~(f European Biochemical Societies, Praha s968, p. 82. 9 J. RENCOV;~, J. I-IuRYCH, J. RosMus AND M. CHVAPIL, Abstr. X I I . Congrrssus Rheumatologicus Internationalis, Praha 2969, abstr. 708. io M. P~NKAL~INEN, I-I. ARO, K. SIMONS AND Ix~. I. I~IVIRIKKO, Biochim. Biophys. Acta, 2_,1 (197 ° ) 559. I I H. P. SARETT AND V. H. CHELDELIN, .]. Biol. Chem., 155 (1944) 153. 12 O. MICKELSEN AND R. S. YAMAMOTO, in D. GLICK, Methods of Biochemical Analysis, Vol. 6, Interscience Publishers, New York, London, 1958, p. 191. 13 S. UDENFRIEND, Fluorescence Assay in Biology and Medicine, Vol. 2, Academic Press, New Y o r k a n d London, 1969, pp. 292-297. 14 O. HAYAISHI, Ann. Rev. Biochem., 38 (1969) 21. 15 I~. E. I~HOADS AND S. UDENFRIEND, Proc. Natl. Acad. Sci. U.S., 60 (1968) 1473. 16 H. R. MAHLER AND ];~. H. CORDES, Biological Chemistry, H a r p e r I n t e r n a t i o n a l ed., J o h n Weatherhill, Inc., Tokyo, 1966, pp. 322-377. 17 M. I¢EKOMKKI, J. JXNNE, E.-L. I4,AHIALA, A. RAINA AND N. RKIHX, Scan& ,]. Clin. Lab. Invest., 23 ,suppl. lO8 (I969) 3° . i8 E.-L. RAHIALA, J. J~,NNE, M. KEKOMAKI, A. RAINA AND N. RXlHX, Biochim. Biophys. Aeta, in the press. I9 R. E. RHOADS AND S. UDENFRIEND, Arch. Biochem. Biophys., 139 (197o) 320.

Biochim. Biophys. Acta, 220 (197 I) 504-508