Clin. Biochem. 9, (6) 256-259 (1976)

Simplified Method for Determination of Protocollagen Proline Hydroxylase NELLY University

of Copenhagen,

BLUMENKRANTZ

and GUSTAV

ASBOE-HANSEN

Department of Dermatology (with Connective Tissue Research ies), Rigshospital, Copenhagen, Denmark

Laborator-

( A c c e p t e d J u l y 22, 1 9 7 6 )

MATERIAL AND METHODS CLBIA, 9, (6) 256-259 (1976)

Clin. Biochem. Blumenkrantz, Nelly and Asboe-Hansen, Gustav

University of Copelzhagen, Dspavt~nent of Dermatology (with Connective Tissue Research Laboratories), Rigshospital, Copenhagen, Dem~mrk SIMPLIFIED METHOD FOR DETERMINATION OF PROTOCOLLAGEN P R O L I N E HYDR OXYL AS E Preparation of ('~C) Pro labeled protocollagen and requirements for its hydroxylation by P P H was investigated. Protocollagen can be prepared by centrifugation of the biological material at 15.009 x g. Substances in current use, viz catalase, bovine serum albumin and dithiothreitol were found unnecessary under the hydroxylation conditions used. A decrease in P P H in the testis and skin of rats with increasing age was demonstrated.

FOR YEARS, HYDROXYPROLINEHAS BEEN USED AS A PARAMETER in studies of collagen biosynthesis. A h i g h cont e n t of h y d r o x y p r o l i n e in th e collagen molecule was the r e a s o n wh y this amino acid was chosen. T h e conditi o n s r e q u i r e d f o r the b i o s y n t h e s i s of h y d r o x y p r o l i n e have been t h e s u b j e c t of m a n y e x p e r i m e n t a l studies. To-day we know t h a t the enzyme involved in t h e hydroxylation, viz protocollagen proline h y d r o x y l a s e ( P P H ) , does not h y d r o x y l a t e f r e e proline. 0 n l y prolyl r e s i d u e s wh i ch have been i n c o r p o r a t e d into protocollagen, a polypeptidic p r e c u r s o r of collagen, or synt h e t i c polypeptides w i t h s i m i l a r a m in o acid sequence ( G l y - P r o - P r o ) ar e h y d r o x y l a t e d by P P H " ~ . Th e enzyme r e q u i r e s ~ - k e t o g l u t u r a t e , ascorbic acid, f e r r o u s ions and a t m o s p h e r i c o x y g e n " ' ~ ' ~ . In a d d i t i o n to t h e m e n t i o n e d co-factors, m o s t a u t h o r s ",s.3.'~ have included in in vitro h y d r o x y l a t i o n s y s t e m s some substances t h e action o f w h ic h is obscure. T h e se substances include d i t h i o t h r e i t o l ( D T T ) , bovine s e r u m a l b u m i n and catalase. W e i n v e s t i g a t e d th e e f f e c t of v a r i o u s c o n c e n t r a t i o n s and lack of co-factors, and s i m i l a r studies w e r e perf o r m e d on the e f f e c t of D T T , bovine s e r u m a l b u m i n and catalase. W i t h t h e assay t h u s elaborated, th e e f f e c t s of a g e i n g on t h e skin and of m a t u r a t i o n on t h e testes of t h e s a m e r a t s w e r e i n v e s t i g a t e d , u s i n g the c o n t e n t of P P H in t h e t i s s u e s as p a r a m e t e r .

Preparation of protocollagen proline hydroxylase. Crude enzyme preparations were extracted from skin and testes of prepubertal, pubertal and adult rats. The body weight was taken as parameter of ageing. Two grams of skin or testes (deprived of albuginea) soaked m 20 ml 0.01 M KCl was homogenised, the former in a Virtis homogeniser at 45,000 x g for 6 minutes, the latter in a Potter homogeniser with teflon pestle at 8,000 rpm for 3 minutes. While working, the homogenisers were surrounded by crushed ice. The homogenates were centrifuged at 15,000 x g for 30 minutes at 4 ° in a Servall centrifuge. The same relationship between weight of tissue and volume of KC1 was kept constant. 0.5 ml of the supernatant was used as enzyme. In some experiments, 0.1, 0.3 or 0.5% Triton was added to preparations of rat skin and testes obtained as indicated above.

Preparation of the unhydroxylated substrate. CsC) Pro labelled protocollagen. Forty-eight 10-day-old chicken embryo tibiae were pre-incubated at 37 ° under a~robic conditions for one hour in 2.7 ml medium of salts, buffer phosphate and glucose c1~. Then 0.3 ml of a 10 mM solution of aa'dipyridyl was added and the pre-incubation continued for further 30 minutes. Immediately a f t e r this, 50 aCi (14C)Pro, diluted in the same medium, was added, and the tissues were incubated under the same conditions for 2 hours. A t the end of the incubation period the tissues were homogenised in the medium, and the homogeniser washed with 3 ml water which was pooled with the hamagenate. The homogenate was then centrifuged at 15,000 x g for 1 hour at 4 ° in a Servall centrifuge. The supernatant was dialysed for 24 hours against 4 changes of 4 1 of 1 M KC1-0.02 M Tris buffer pH 7.6. After dialysis, the sample was boiled for 5 minutes, and, after cooling, an aliquot was counted in a liquid scintillation cuunter. The sample was diluted with 1 mM KCI to give 80,000-100,000 dpm/ml. One ml aliquots were distributed and stored frozen in 20 ml ampoules. Preparation of co-factors. A solution of co-factors was prepared freshly before the hydroxylation assay. The cofactors' solution was made up of solution A: 111.2 mg FeS04 in 10 ml water; and solution B: 29.2 mg a-ketoglutarate and 140,8 ascorbic acid dissolved in 30 ml water. 0.5 ml of solution A was mixed with 30 ml of solution B and 10 ml 0.5M Tris-HC1 buffer pH 7.8 at 25 ° . 0.8 ml of this solution was used as the co-factors' solution in the hydroxylation procedure. The content of co-factors was: a-ketoglutarate 0.5 raM, ascorbic acid 2.0 mM, ferrous sulphate 0.05 raM. Enzyme assay. The enzyme assay was performed directly in the very ampoules in which the substrate was stored after being defrosted. One mi enzyme and 0.8 ml of the co-factors' solution was added to 1 ml substrate (present in the ampoules). Under shaking, the samples were incubated for 1 hour at 37 °. The reaction was stopped either by addition of an equal volume of concentrated HC1 and hydrolysis of the samples or by addition of 15 ml cold acetone allowing the proteins to precipitate. Thereafter, the samnles were centrifuged at 2000 rnm for 30 minutes.

DETERMINING PPH

257

The supernatant was discarded and the pellet hydrolysed with 2 ml of 6 N HCl. Hydrolysis was performed at l l 0 ° C for 16-18 hours. Thereafter, the HCI was evaporated under vacuum at 65 °. The samples were suspended in 5 ml buffer and 2 aliquots separated. One aliquot of 0.1 ml was used to determine the total '~C while another 4.5 ml aliquot was used to determine the (J4C)Hyp formed. (~4C)Hyp was assayed according to procedure IIB of Blumenkrantz and Asboe-Hansen{~L

e n c e s in p e r c e n t a g e h y d r o x y l a t i o n w e r e seen, w h e t h e r the 15,000 x g or t h e 100,000 x g s u p e r n a t a n t w a s u s e d as s u b s t r a t e . T h e b l a n k s o f e a c h one, r u n w i t h w a t e r i n s t e a d o f enzyme, w e r e also s i m i l a r . T h e d i a l y s e d m e d i u m (in w h i c h the b o n e s w e r e inc u b a t e d f o r p r e p a r a t i o n of t h e p r o t o c o l l a g e n ) , w h e n u s e d as s u b s t r a t e , s h o w e d s l i g h t l y h i g h e r v a l u e s t h a n t h e blanks.

Experimental. We tried to use the incubation medium (see Preparation of unhydroxylated Substrate) as substrate for PPH after dialysis under the same conditions as indicated for the homogenised tissues. As most authors have used the supernatant after centrifugation at 100,000 x g as substrate, a comparison was made between this and our substrate. The effect of decreasing concentrations of each of the co-factors on the hydroxylation of (~4C)Pro labelled protocollagen was studied. The concentrations assayed were 1) normal (as indicated above) or 2) 1:2, 3) 1:4; 4) 1:8; and 5) 0. Similar studies were performed on decreasing concentrations of the mixture of co-factors.

o f a c h r o m o g e n w h e n F e 2+ a n d a s c o r b i c a c i d a r e mixed. A c h r o m o g e n w a s p r o d u c e d w h e n F e ~+ and a s c o r b i c acid w e r e m i x e d a t a p H r a n g i n g f r o m 5.0 to 8.1. T h e p e a k o f k - m a x of t h i s c h r o m o g e n w a s 540 nm. T h e c h r o m o g e n d i s a p p e a r e d soon, i.e. a f t e r 10 m i n u t e s ' i n c u b a t i o n , in the a b s e n c e o f t h e e n z y m e . In the p r e s e n c e of t h e enzyme, t h e c h r o m o g e n r e m a i n e d e v e n a f t e r 1 hour, i.e. a t t h e e n d o f the i n c u b a t i o n a t 37 °. T h e n a t u r e o f t h e c h r o m o g e n is u n k n o w n .

Comparison of PPH activity in crude extracts of rat kidney and testis. Pools of rat kidneys weighing 150 mg were homogenised in 25 ml 0.15 N NaC1, 0.01 N KCl or 0.1 N acetic acid. Pools of rat testes weighing 150 mg were homogenised in 25 ml water, 0.15 N NaC1, 0.01 KC1 or 0.1 N acetic acid. Homogenisation was performed in a teflon pestle homogeniser. A f t e r - h o m o g e n i s a t i o n , the samples were centrifuged in a Servall centrifuge as indicated in "Preparation of the unhydroxylated substrate". One ml of the supernatant was used as source of enzyme for the in vitro hydroxylation of protocollagen.

E x t r a c t i o n o f c r u d e enzyme. C o m p a r i s o n o f t h e P P H e x t r a c t e d f r o m r a t kidney w i t h the solutions of NaCI, KCI or a c e t i c a c i d s h o w e d t h e f o l l o w i n g h y d r o x y l a t i o n s : 1.87, 1.24 a n d 0% r e s p e c t i v e l y . A s to P P H f r o m testis, t h e % h y d r o x y l a t i o n w a s 5.70, 3.87, 5.02 and 0 f o r t h e en;zyme e x t r a c t e d w i t h w a t e r or the s o l u t i o n s o f NaCI, KC1 a n d ace.tic acid, r e spectively.

E f f e c t of a g e on the content of PPH in rat skin and testis.

Production

E f f e c t of c o n c e n t r a t i o n of c o - f a c t o r s on t h e h y d r o x y lation of protocollagen.

Crude enzyme preparations of prepubertal, pubertal and adult rat skin and testis were obtained and assayed as indicated in "Enzyme assay". Two grams of r a t skin were homogenised in 20 ml 0.01 N KC1. The same proportion of the weight of testicular tissue and volume of 0.01 N KC1 were kept for the testicular preparation. {In case of lower weight, the volume was reduced proportionally).

Ascorbic acid r~nd c~-ketoglutc~ra~e. T h e r e was no s t r i k -

Calculation. The percentage hydroxylation was calculated as follows:

Ferro~as ions. N e i t h e r an i n c r e a s e of the c o n c e n t r a t i o n

dpm ('4C) Hy~ found dpm total ~4C in substrate

X 100 = % hydroxylation

As, in all experiments, the wet weight of the tissues used as source of enzyme was the same, the results were percentage hydroxylation of protocollagen by 100 mg wet weight of tissue.

i n g r e d u c t i o n o f the h y d r o x y l a t i o n by a r e d u c t i o n to ~ of the c o n c e n t r a t i o n of ascorbic acid or a - k e t o g l u t a r a t e . N e v e r t h e l e s s , both s u b s t a n c e s w e r e n e c e s s a r y f o r the h y d r o x y l a t i o n as c o m p a r e d w i t h the s h a r p d e c r e a s e o b s e r v e d in the a b s e n c e of e a c h one o f the two (Table 1). of F e 2+ by 2 or 3 times, n o r a r e d u c t i o n to 1/~ o r 1/~ r e s u l t e d in a n y d i f f e r e n c e in t h e l e v e l s o f h y d r o x y l a tion of p r o t o c o l l a g e n in c o m p a r i s o n to t h e 0.05 ml~I ( n o r m a l c o n c e n t r a t i o n ) . T h e r e d u c t i o n of t h e hyd r o x y l a t i o n o b s e r v e d in the a b s e n c e o f F e 2+ w a s less m a r k e d t h a n t h a t o c c u r r i n g in the a b s e n c e of a s c o r bic acid or a - k e t o g l u t a r a t e ( T a b l e 1).

M i x t u r e of co-factors. The r e d u c t i o n to 1/s or f u r t h e r

RESULTS C o m p a r i s o n of s u b s t r a t e s p r e p a r e d b y c e n t r i f u g a t i o n at 15,000 x g and 100,000 x g. No s i g n i f i c a n t d i f f e r -

on o f the a m o u n t of the c o - f a c t o r s ' m i x t u r e p r o duced a r e m a r k a b l e d e c r e a s e of the h y d r o x y l a t i o n of p r o t o c o l l a g e n ( T a b l e 1).

TABLE 1 EFFECT OF DIFFERENT CONCENTRATIONSOF CO-FACTORSON THE HYDROXYLATIONOF (t4C) PRO

LABELLEDPROTOCOLLAGENBY PPH.

mM 2.0 (N) 1.0 0.5 0.25 None

% hydrox.

mM

% hydrox.

7.20 6.69 6.61 6.12 0.87

0.25 (N) 0.125 0.0625 None

9.02 9.25 6.06 0.67

Mixture of co-factors

FeS04

=-ketoglutarate

Ascorbic acid

mM 0.15 0.10 0.05 (N) 0.025 0.0125 None

Skin of rats of different age were used for the preparation used in each experiment. N ffi normal (as indicated in Material and Methods). % hydrox. = percentage hydroxylation.

% hydrox, 16.16 17.12 17.04 17.89 18.25 4.05

± q4444-

1.2 1.1 1.2 1.5 1.7 0.5

Amount N 1/2 1/4 1/8 1/16

% hydrox. 14.87 14.36 13.55 10.18 3.70

258

BLUMENKRANTZ AND ASBOE-HANSEN TABLE 2 EFFECT OF DECREASE OR ABSENCE OF BOVINE SERUM ALBUMIN, CATALASE AND DTT

ON THE IN VITRO HYDROXYLATIONOF (~4C) PRO LABELLEDPROTOCOLLAGEN.

Bovine serum albumin Amount % hydrox.

Amount

N 1/2 1/4 1/8 1/10 None

N 1/2 1/4 1/8 None

8.40 8.50 8.38 9.50 7.75 7.96

± 0.6 =t= 0.9 ~- 0.8 ± 1.0 4- 1.0 ± 1.0

Catalase

% hydrox.

Amount

18.06 18.41 17.77 17.94 17.76

N None

-4- 1.0 ± 1.0 d- 0.9 -~ 0.9 ± 0.9

DTT

% hydrox.

12.0 + 0.6 12.1 d- 0.5

Results represent average of 2 experiments. N = normal. hydrox. = percentage hydroxylation.

E f f e c t of different concentrations of bovine s e r u m albumin (BSA) on the hydroxylation of protocollagen by P P H . N o s i g n i f i c a n t d i f f e r e n c e s in the hydroxylation was observed by decrease or withd r a w a l of BSA (Table 2). E f f e c t of the p r e s e n c e or a b s e n c e of DTT on the enz y m a t i c hydroxylation of C ' C ) P r o l a b e l e d protocoll a g e n by P P H . No e f f e c t on the p e r c e n t a g e h y d r o x y l'ation was observed by w i t h d r a w a l of DTT from the h y d r o x y l a t i o n s y s t e m (Table 2). E f f e c t of decrease or withdrawal of c a t a l a s e . No e f f e c t was noticed on the levels of h y d r o x y l a t i o n of p r o t o c o l l a g e n by P P H when the a m o u n t of c a t a l a s e was d e c r e a s e d or even w i t h d r a w n f r o m the h y d r o x y lation ~ystem ( T a b l e 2). E f f e c t of Triton X-100 on P P H . A d d i t i o n of i n c r e a s i n g c o n c e n t r a t i o n s of T r i t o n X-100 (0.1%, 0.3% or 0.5%) to the c r u d e enzyme p r e p a r a t i o n f r o m r a t skin showed no s i g n i f i c a n t e f f e c t on the h y d r o x y l a tion of p r o t o c o l l a g e n by P P H . E f f e c t of different a m o u n t s of enzyme h y d r o x y l a tion, and comparison of d i r e c t h y d r o l y s i s w i t h hydrolysis a f t e r p r e c i p i t a t i o n with cold acetone of the h y d r o x y l a t i o n product. An i n c r e a s e in p e r c e n t a g e h y d r o x y l a t i o n was noticed a f t e r p r e c i p i t a t i o n and when the a m o u n t of enzyme was i n c r e a s e d (Table 3).

20

I

08

.

16

.

.

-

r I I:-.

.

-

1

_

Ii

i S ~



SKIN

PPH



TESTIS

G

2 q

~

~'0

,

i

PPH

] i g

80 BODY

I00 WEIGHT

19'0

i

i

140

160

Fig. I ~ Pro~ocollagen proHne hydroxylas¢ eztracted from skin and ~es~s of ~a~s of d~fferen$ weights as assayed on ("C) Pro-labeled eubetrate. Note decreased enzymatic ae~i~i~y ~ i t h ~ncreasing welght.

TABLE 3 EFFECT OF DIFFERENT VOLUMES OF THE C R U D E E N Z Y M E ON HYDROXYLATION. DIRECT HYDROLYSIS AND HYDROLYSIS AFTER PRECIPITATION WITH ACETONE.

Sample Substrate -{co-factors plus

Direct hydrolysis % hydrox.

0.5 ml enzyme 1.0 ml enzyme 1.5 ml enzyme

14.26 15.91 17.09

Hydrolysis after precipitation % hydrox. 17.13 21.06 24.84

E f f e c t of a g e on the content of P P H in testis and s k i n of r a t s . D e c r e a s i n g P P H p e r s i m i l a r w e i g h t of skin and t e s t i s was noticed in r e l a t i o n to age. In y o u n g e r r a t s the a c t i v i t y of P P H was h i g h e r t h a n in m a t u r e a n i m a l s (Fig. 1). DISCUSSION The elevated content of P P H in skin and t e s t i s of younger r a t s is noteworthy and unexplained. Other authors have found differences in the content of P P H in relation to embryonic m a t u r a t i o n Ce). The s t a b l e P P H a c t i v i t y with the w i t h d r a w a l of DTT is s u r p r i s i n g if one considers t h a t many authors indicate the need of D T T " ' ~' 3,.,. In o r g a n c u l t u r e of chicken embryo we found a decreased h y d r o x y l a t i o n of C4C)Pro and C4C)Lys in the presence of increasing doses of DTT. Our results f r o m the organ culture system are in a g r e e m e n t with the statements of Popenoe et al "), in the sense t h a t DTT affects P P H activity-directly. According to the mentioned authors, i n c u b a t i o n of the enzyme with DTT d e s t r o y s the enzyme activity in a few hours. As to catalase, o u r results are in agreement with the fact t h a t catalase is inhibited by ascorbic acid, as reported by e r r c". P e t e r k o f s k y and DiBlasio '3~ s t u d y i n g the requirements for t r i t i a t e d w a t e r release d u r i n g the hydroxylation of protocollagen, used a crude p r e p a r a t i o n and observed that, in the absence of a - k e t o g l u t a r a t e or ascorbic acid, the a c t i v i t y was about 2..5%. In the absence of F e z+, catalase, DTT or bovine serum albumin, the activity was 45, 93.4, 74.5 and 26%, respectively. In our system we observed the same p a t t e r n except for bovine serum albumin, catalase and DTT. The difference f o r bovine serum albumin m i g h t be related to the albumin p r e p a r a t i o n . Popenoe et al "> studied the effect of various albumin p r e p a r a t i o n s on chick embryo P P H , as measured by the release of t r i t i a t e d w a t e r into the hydroxylation system. I n

DETERMINING

the absence of the enzyme the authors found an appreciable release of SH from the labeled protocollagen into the medium a f t e r 30 minutes of incubation in the presence of undialysed albumin. This effect was not observed in the presence of dialysed albumin, in which case it was proportional to the amount of the enzyme. R~IFERENCES 1. Prockop, D. J. and Juva, K. (1965). Proc. nat. Acad. Sci. (Wash.) 53, 661-668. 2. Kivirikko, K. I. and Prockop, D. J. (1967). Proc. nat. Acad. Sci. (Wash.) 67, 782-789.

PPH

259

3. Peterkofsky, B. and DiBlasio, R. (1975). Anelytr.-Biochem. 66, 279-286. 4. Harwood, R., Grant, M. E. and Jackson, D. S. (1974) Biochem. J. 144, 123-180. 5. Blumenkrantz, N. and Asboe-Hansen, G. "(1975). Analyt. Biochem. 66, 330-839. 6. Blumenkrantz, N. and Asboe-Hansen, G. (1972). Mech. Ageing Dev. 1, 445-450. 7. Popenoe, E. A., Aronson, R. B. and Van Slyke, D. (1969). Arch. Biochem. 133, 286-292. 8. Orr, C. W. M. (1969). Biochem. biophys. Res. Commun. 23, 854-

Simplified method for determination of protocollagen proline hydroxylase.

Clin. Biochem. 9, (6) 256-259 (1976) Simplified Method for Determination of Protocollagen Proline Hydroxylase NELLY University of Copenhagen, BLUME...
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