Mechanism of the Action of Chelating Agents on Proline Hydroxylation and its Incorporation into Collagenous and non-collagenous Proteins

Transcription

1 European J. Biochem. 2 (1967) Mechanism of the Action of Chelating Agents on Proline Hydroxylation and its Incorporation into Collagenous and non-collagenous Proteins M. CHVAPIL, J. HURYCH, l3. EHRLICHOV~, and M. TICHP Department of Experimental Biology, Institute of Industrial Hygiene and Occupational Diseases, Prague (Received April 28, 1967) The effect of concentration of some chelating agents on proline hydroxylation and the synthesis of collagenous and non-collagenous proteins was studied in skin slices of chick embryo. In a group of eight chelating agents a good correlation was found between the degree of inhibition of hydroxylation and the stability constant of ferrous chelates. No correlation with the stability of cupric complexes was found. The failure of 2,9-dimethyl- 1,iO-phenanthroline to inhibit hydroxylation also suggests participation of Fez'. The study of the influence of different times of preincubation of the tissue with chelating agents showed that 2,2'-dipyridyl and 1,lo-phenanthroline inhibit the hydroxylation of proline already within 1 minutes. In addition 2,2'-dipyridyl in 1 mm concentration also inhibits after 2 hours of action the incorporation of [14C]proline into collagenous proteins and does not change during a 4 hour incubation t'he synthesis of non-collagenous proteins. Phenanthroline, in the same concentration, inhibits already during the first 6 minutes of incubation both the synthesis of collagen as well as of non-collagenous proteins. Chelating agents which do not penetrate cell membrane are without effect on the reactions studied. The blockade of hydroxylation is a specific result of the chelation of bivalent iron. Chelating agents influence more easily the incorporation of amino acids into collagenous proteins than synthesis of globular proteins. This effect is considered specific because it is related to the chelating properties of the substances investigated. All other investigated substances without chelating properties inhibit, according to the concentration used, synthesis of both types of protein equally, so that the decrease in hydroxyproline formation is secondary to the inhibition of proline incorporation. Close correlation between the inhibition of protein synthesis and general toxicity of these substances has been found. Previous publications [l,2] from this Laboratory have shown that 2,2'-dipyridyl at mm concentrations blocks hydroxylation of collagenous proline. In contrast, 1,lO-phenanthroline at.1 to.5 mm concentrations blocks hydroxylation, too, and in addition, after 2 hours of incubation, partially inhibits the incorporation of all amino acids into collagen. However, the synthesis of non-collagenous proteins in chick embryo skin slices is not modified. Some other chelating agents differed markedly in their capacity to inhibit on the one hand hydroxylation and on the other the incorporation of proline into proteins. Isomers of phenanthroline and of dipyridyl, without chelating properties, do not affect collagen hydroxylation either, but they inhibit to the same extent proline incorporation into both collagenous and non-collagenous proteins of the skin [3]. Non-Standard Abbreviations. Cysteine, CySH; diethyidithiocarbamate (sodium salt), DDC; 2,2'-dipyridyl, Dip; 8-hydroxyquinoline, HYQ; D-penicillamine, Pen; 1,lOphenanthroline, Phen; ethylenediaminotetraacetic acid disodium salt, EDTA. The present study was undertaken to explain the varying ability of chelating agents to inhibit proline hydroxylation and to find an explanation for the inhibition of incorporation of amino acids into collagenous proteins by phenanthroline, found previously. For this reason we studied the relation between the concentration, time of preincubation of skin slices with chelating agents and between some substituted phenanthrolines and the three reactions mentioned above. MATERIAL AND METHODS The same experimental procedure as described in previous papers was used [l, g of approx. 12 days old chick embryo skin slices were incubated for 2 hours with [l4c]proline (5 $3) at 37" in a 1 ml Krebs-Ringer bicarbonate buffer, ph 7.4, in oxygen atmosphere ( + 5 O/, [vlv] CO,). The samples containing appropriate concentrations of the chelating agent to be investigated were compared with control samples. Fractions of collagenous and non-collagenous proteins soluble in.2 M NaCl were removed and the

2 23 Chelating Agents and Collagen Biosynthesis European J. Biochem. residual collagenous proteins extracted according to the method of Fitch et al. [4]. The non-collagenous residue was washed with alcohol, alcohol-ether (1 :4, v/v) and ether. In the hydrolysate, proline was determined according to Troll and Lindsley [5] and hydroxyproline according to Stegemann [el. Radioactive proline and hydroxyproline were isolated according to Peterkofsky and Prockop [7]. Radioactive assay was performed on a Packard Tri-Carb Liquid Scintillation Spectrometer Model The counting efficiency was about 72,lo. Experiments to clarify the rate of action of the selected chelating agents were carried out as follows : Skin slices (I g wet weight) were incubated with mild shaking for different times, ranging from 1-24 minutes, in a Krebs-Ringer bicarbonate buffer (5ml) with a 1 mm concentration of chelating agent; control samples were incubated with buffer only. Afterwards the slices were separated by centrifugation and washed quickly three times in a salt solution. The incubation with [14C]proline (3.82 $/flask) was made in the usual way and lasted 1 hour. The collagenous and non-collagenous proteins were isolated as described above. The substituted phenanthrolines (Roch-Light- Laboratories, Colnbrook England) and 2,2'-dipyridyl, 1,lo-phenanthroline and sodium diethyldithiocarbamate (Merck, Darmstadt, West Germany) were of p. a. purity. The origin and purity of other compounds were given in our previous communication [3]. The second procedure used for characterization of general toxicity of individual substances studied was the inhibition of movement of worms Tubifex tubifex [8]. The data refer to the concentration of an investigated substance at which the incessant movement of all worms was inhibited within 3 seconds after their immersion into a solution of the substance at a temperature of 2". The values are given as relative toxicity related to the toxicity of NaN, as reference substance. For the calculation of quantum chemical data the simple Huckel's molecular orbital method [9] was used. The meaning of the individual symbols and the parameters used are given in the respective Table. RESULTS AND DISSCUSSION The Effect of Concentration of a Chelating Agent The effect of 2,2'-dipyridyl and 1,lO-phenanthroline in a concentration range of 1 mm-1 pm, of diethyldithiocarbamate and 8-hydroxyquinoline (5.-.1 mm) and of D-penicillamine (1-.1 mm) on the formation of [14C] hydroxyproline in collagen and on the incorporation of [14C] proline into collagenous and non-collagenous proteins was studied. When comparing the investigated substances accord- ing to concentrations which cause more than a 8/, inhibition of proline hydroxylation (Fig. 1) we obtain the order Phen > Dip > HYQ = DDC > Pen. It is evident that the effective inhibitory concentration among individual chelating agents differs always by approximately three orders. Similarly, concentrations were found in which chelating agents do not influence, after 2 hours of action on skin slices, the process of hydroxylation : 1,lo-phenanthroline is ineffective in 1-6M concentration, 2,2'-dipyridyl and 8-hydroxyquinoline in 1-5M concentration and D-penicillamine does not act in M concentration (Fig. 1). I z y 1 - s - W 2 i 8- E > - : % 4- >- k - 2 I- : - LL a cn 2- i. i - - CONCENTRATION (-LOG [ti) Fig. 1. The effect of concentration of investigated chelating agents on the hydroxylation of collagenous proline. Estimated in residual collagen after extraction with.2 M NaCI. Specific activity of hydroxyproline expressed in per cent of specific activity of control samples ( 1 /, corresponds to 12,4 counts/min per pmole hydroxyproline) When proline hydroxylation is blocked by chelating agent, an atypical collagen is formed which does not contain hydroxyproline and hydroxylysine. After incubation with dipyridyl (1 mm) the specific activity of [14C] proline in collagen is twice that of controls. With 1,lO-phenanthroline (.1 mm) the specific activity is the same as in the controls. We have proved earlier [lo] that in both cases hydroxyproline-deficient collagen contains the proline radioactivity which is the sum of the actual collagenous proline and of proline to be hydroxylated. Phenanthroline in.1 mm concentration totally blocks hydroxylation and inhibits simultaneously by approximately 5 /, the incorporation of all amino I

3 Vol. 2, No. 2, 167 N. CHVAPIL, J. HURYCH, E. EFIRLICHOV~, and M. TICH~ 231 acids, including proline, into the collagenous molecule. Further on we refer in Figures and Tables to the sum of radioactivities of both imino acids. Let us express changes in the specific activity of experimental samples in per cent of the specific activity of control samples. The results prove that in a.1-1 mm concentration 2,2'-dipyridyl, after a 2 hour incubation, has no influence on the incorporation of the sum of >- W a cn NONCOLLAGENOUS 114C1 HYQ Phen5\ \ ' h L CONCENTRATiON (-LOGrMI) Fig. 2. The effect of concentration of investigated chelating agents on the incorporation of proline into collagenous and noncollagenous proteins. Collagenous protein expressed as the sum of imino acids (specific activities of control sample are 12,4 counts/min per pmole hydroxyproline and 13,5 counts/min per pmole proline), non-collagenous proteins by means of incorporated proline (1 O/, equals to 51, counts/ min per pmole proline) imino acids into collagen. On the other hand, 1,IOphenanthroline, which inhibits effectively the hydroxylation already in.1 mm concentrations (Fig. l), decreases the radioactivity of imino acids in collagen to 5 /, and more in the range of.5 to 1 mm concentration (upper part of Fig.2). With regards to the effect of various concentrations of both chelating agents on proline-specific activity in non-collagenous proteins (lower part of Fig.2) compared with their effect on the sum of [W] imino acids radioactivity in collagen (upper part of Fig.2) there is a stimulation of synthesis of! 7 non-collagenous proteins after dipgridyl, in the range of. I - 1 mm concentrations, whereas collagen synthesis is not affected. We encountered this phenomenon already in previous works [2]. Since, however, there is an increase higher than 2 /,, of the control value which can be reproduced, we do not consider it as coincidental. It may be related to removal of some metals which slightly inhibit synthesis of globular proteins from incubating mcdium. As far as the effect of 8-hydroxyquinoline is concerned its capacity to inhibit proline hydroxylation is less than that of 2,2'-dipyridyl (Fig.1). On the other hand, this chelating agent affects incorporation of proline into both types of proteins to the same extent and more efficiently than dipyridyl (Fig. 2). This favours the hypothesis that this effect is related not to chelating properties of S-hydroxyquinoline but to some other reactivities of its molecule. This experiment does not explain the puzzling phenomenon that I,lo-phenanthroline, in concentrations of.5-.1 mm, has no influence on the incorporation of proline into non-collagenous proteins, whereas it decreases simultaneously the incorporation of imino acids into collagen by 5 /,. Only in higher concentrations of 1,lo-phenanthroline (.5-1 mm) is the incorporation of proline into both types of the investigated protein inhibited. In any case, these results suggest the possibility to influence independently the biosynthesis of collagenous and non-collagenous proteins by chelating agents, and disclose a differential susceptibility of synthetic pathways for collagenous and non-collagenous proteins towards the action of chelating agents. The Effect of Chelating Agents not Permeating Cell Menabrane It is well known that some chelating agents do not penetrate the cell membrane so that their effect is limited to the extracellular distribution space [l 11. Among these are EDTA, Tiron or 8-hydroxyquinoline- 5-sulfonic acid, which did not affect in our experiments the three reactions studied in skin slices system (Table 1). Thus intracellular distribution of a chelating agent is indispensable for its proline hydroxylation inhibiting activity and its effect on protein synthesis. The Effect of Different Times of Preincubation of Tissue with Chehtiizg Agents Previous experiments have shown a relationship between the degree of the inhibition of proline hydroxylation and the stability constant of chelating agents with bivalent iron. The efficiency of chelating agents is also related to its ability to penetrate cellular membranes insofar as their effect must be intracellular, since proline hydroxylase has been

4 ~ ~~~~ 232 Chelating Agents and Collagen Biosynthesis European J. Biochem. Table 1. The evidence on the ineffectiveness of chelating agents not penetrating the cell membrane on proline hydroxylation and its incorporation into proteins Owing to the low solubility of 8-HYQ-sulfonic acid in aqueous medium a mixture of ethanol-water (1: 1, v/v) was used and compared with corresponding control samples. Skin slices incubated for 2 hours under standard conditions. Compare the results with Fig. 1 where the activity of non-substituted 8-HYQ is presented. Concentration of chelating agents = 1 mil1 Chelating agents Incorporation into collagen ["C] hydroxyproline ["GI prolinc x counts/min/pmole Incorporation of [14C] proline into non-collagenous proteins of controls) Control (aqueous) 11.3 (1) 12.3 (1) 47.8 (1) EDTA 1.7 (95) 12.5 (12) 41. ( 86) Pyrocatechol-3,5-disulfonic acid (Tiron) 9.6 (85) 14.8 (12) 55. (115) Control (ethanolic) 9.1 (1) 1.1 (1) 42. (1) 8-hydroxyquinoline-5-sulfonic acid (sodium) 9. (99) 14.6 (119) 47.5 (113).--.--I" dipyridyl block proline hydroxylation already after 1 minutes of incubation (Fig.3). The shape of the 2 8 curves for phenanthroline and dipyridyl shows a slightly higher rate of action for phenanthroline. But 1 HYDROXYPROLINE Li4C1 this difference cannot be the cause for their different A 14 A effect because the incubation with labelled amino A acids lasted, in all our previous experiments, for ':O z _I E o L P 3 Lbo-e--i h w min minutes during which this initial differences in the rate of penetration of the substance into the cell L L, I would be totally obscured. If I mm phenanthroline blocks proline hydroxylation completely already within 1 minutes (upper part of Fig. 3) then it decreases the specific activity of [14C] imino acids in collagenous (middle part of Fig. 3) and in non-collagenous proteins (lower part of Fig. 3) u- u only by about 6 /,. As had been shown (Fig.2) min 24 w phenanthroline in concentrations of mm n I had no effect on the synthesis on non-collagenous A proteins after 2 hours of incubation, whereas it Pen \: inhibited the formation of collagenous proteins within the same time approximately by 5 /,. The NONCOLLAGENOUS [14C1 difference in the effect of phenanthroline depends on, Phen PROLINE] \- the local concentration of the agent, which is dependent not only on the dose but also on the duration rnin 24 of action. The concentration used in this experiment Fig. 3. The rate of action of investigated chelating agents on the hydroxylation of proline and its incorporation into collagenous was too high so that the differences of the action on and non-collagenous proteins. Skin slices of chick embryo were collagenous and non-collagenous proteins could not preincubated at 37" with chelating agents for different times. be detected. After washing, the slices were further incubated 1 hour with [I4C] proline. Details under the section Material and Methods The validity of this conclusion is also substantiated by the time-course of incorporation of [14C] imino acids into collagen isolated from tissues incushown to be localized in the fraction of soluble cyto- bated with 1 mm dipyridyl (middle part of Fig.3). plasmic proteins (15, xg supernatant) [12,13,14]. During the first 12 minutes of the action of dipy- In order to elucidate the rate of the action of ridyl no influence on the synthesis of collagenous dipyridyl, phenanthroline and D-penicillamine we proteins could be found. This was in good agreement preincubated the skin slices for different periods with our previous experience where the incubation (from 1 minutes to 4 hours) in equimolar concen- always lasted for 12 minutes [I]. But during a trations (1 mm) of the substances mentioned. We longer action of dipyridyl, lasting 4 hours, the incorthen incubated the tissue for a further 6 minutes poration of proline into collagen was also inhibited, in a labelled proline medium. The control tissue was as was the case with phenanthroline. After the same incubated in a similar way, though without chelating time of dipyridyl action, however, the synthesis of agents. non-collagenous proteins still does not change (lower The time-course of incorporation of [14C] proline part offig. 3). A really high concentration of dipyridyl into collagen is given in Fig.3. Phenanthroline and (1 mm) markedly inhibits within 2 hours the activ-

5 ~~~ ~ Vol.2, No.2, 1967 M. CHVAPIL, J. HURYCH, E. EHRLICHOV~, and M. TICH~ 233 ity of proline in both types of proteins studied (Fig. 2). Penicillamine had no effect in 1 mm concentrations for up to 2 hours [3]. After 4 hours of incubation the hydroxylation of proline and incorporation of imino acids into collagen decreased approximately in the same way, whereas the synthesis of non-collagenous proteins remained unchanged. The Effect of Xubstituted Phenanthrolines It is known, that methyl- or phenyl-substituted phenanthrolines penetrate more easily through the cell membrane than the parent molecule [15]. Also the stability constants with Fez+, determined either The finding that 2,9-dimethyl-1,lo-phenanthroline does not inhibit proline hydroxylation is a further proof that Fe2+ and not Cu2+ participates in the hydroxylating system. The relatively bulky methyl group in the neighbourhood of the active chelating center forms a steric hindrance to the fornmtion of a stable complex which has not the possibility of binding metal cations of a small radius. For this reason the binding of the ferrous ions (radius.83a) does not occur, whereas larger copper ions (radius.96 A) are well bound [15]. It was proved in model experiments in vitro that ferrous, cuprous or trivalent chromium ions may play a role in proline hydroxylation [17]. We therefore compared the stability of the complex of chelat- Table 2. The relation between the reactivity of substituted 1,lO-phenanthrolines and their effect on hydroxylation and incorporation of (14C] proline into proteins Effective concentration = 1V 31 - Effective concentration = 1F M Incorporation into collagen Incorporation Incorporation into collagen Incorporation into non- into non- Compound log BaC kp Q N ~ [lpci collagenous collagenous Hydroxy- ["C] Proline ~4c1 I,"cl proteins Proline Hydroryproline proline ["GI Proline proteins pc1 Proline x counts/min/pmole of controls) Control ) 15.9 (1) 48. (1) 9.8 (1) 17.2 (1) 53.8 (1) 1,lO-phenanthroline ) 23.7 (149) 48.9 (12).7 ( 7) 3.8 (22) 81.7 (115) 5,6-dimethyl-l,lO-phen ) 8.4 ( 53) 25. ( 52).6 ( 6) 3. (175) 53.7 (1) 4,7-diphenyl-l,lO-phen ) 13.7 ( 86) 36.5 ( 76) 2.9 ( 3) 29. (168) 63 ( 43) 5-nitro-lJO-phen ) 32.1 (22) 58. (121) 9.7 (1) 23.1 (134) 49.8 ( 93) 2,9-dimethyl-l,lO-phen ) 18. (133) 53.7 (112) 9.1 ( 93) 21. (122) 58.3 (18) (kd is the energy of the highest occupied (lowest free) n-molecular orbital, parameters of the heterocyclic nitrogen: 6~ = 1.5, PCN =.8; of the nitro group 6~ = 2, 6 = 1, QCN =.7; the hyperconjugation of the methyl group was considcred. Quantities 6, a P, are defined as follovs: a, = a + 8, B, Ow = e, 19, where a, and Bw stand for coulomb and resonance integrals. k, is a magnitude proportional to the first n-electron ionization potential and is a measure of the oxidisability of the wholc system. The characteristic k4 is a measure of the n-electron affinity and characterizes the reducibility of the whole system. Q.v is the sum of the n-electron densities for both nitrogen atoms. Data in parenthesis refer to residual activity presented as percent of control value. C log is log of stability contants (8. = K1 x K, x Ii,, where Kl, Ks and Ka are step stability constants). directly or suggested according to the calculated density of n-electron on nitrogen atom (Table 2), are higher for these derivates than the phenanthroline itself. Only the nitro derivate forms a less stable complex with iron than phenanthroline itself [16]. These properties as well as their oxidisability and reducibility were compared with the effect on the three phenomena studied. In agreement with findings mentioned previously in this study it was shown that substances forming more stable complexes with ferrous ions are generally more effective. This applies to methyl and phenyl phenanthroline derivates which in.1 and.1 mm concentrations inhibit, just like 1,lo-phenanthroline, almost completely the hydroxylation, but in addition, after a 2 hour action, they inhibit more markedly the incorporation of amino acids into collagenous as well as into non-collagenous proteins (Table 2). ing agents mentioned with Fez+ or Cu2+ with their capacity to inhibit proline hydroxylation. Only with bivalent iron complexes was a very good correlation found between the inhibition of hydroxylation and the stability constant of the complex (Fig.4). These experiments also show that each of the phenomena studied (i. e. proline hydroxylation, incorporation of proline into both groups of proteins) is of a different susceptibility to the effect of chelating agents. The most sensitive reaction is proline hydroxylation which is always inhibited by different chelating agents at low concentrat,ions and within a very short time. The least sensitive reaction to the effect of chelating agents is the synthesis of noncollagenous proteins. Their synthesis could be inhibited in the biological system used only at relatively high concentrations. The effect on the synthesis of collagenous proteins which seemed at the

6 234 Chelating Agents and Collagen Biosynthesis European J. Biochem.,/5.6-DI 'hen M ETHYL-/ Phei I beginning to be specific only for the effect of phenanthroline, can be evoked also by dipyridyl and probably by further iron complexing agents, the effectiveness of which depends on their local concentration in the cell. / I /Pen CySH, I, LOG p n Fig. 4. Relation between stability constants of different chelatiq agents with bivalent iron and their efficiency to inhibit proline hydroxylation. Efficiency is expressed as log of minimum concentration which causes a greater inhibition of proline hydroxylation than 8 /,. Log pn are log of stability constants, Pn = Kl x K,... x K,,, where K are step stability constants The Relation of the Effect to the General Toxicity A further ana1ys;s of the connection between the sequential reactions mentioned above and the properties of the substances studied showed that substances without chelating properties cause the same degree of inhibition of proline incorporation into both collagenous and non-collagenous proteins. In this way they lower indirectly the amount of the hydroxyproline in collagen, because the hydroxylation occurs after the proline incorporation into the polypeptide [13]. It cannot be presumed that such a heterogenous group of substances given in Table 3 inhibits protein synthesis always by the same mechanism. But in many of these substances good correlation may be found between their general toxicity and the ability to inhibit protein synthesis. The toxicity is represented by ED,,, which causes stopping of the movement of worms Tubifex tubifex. On the contrary, no Table 3. The relation between general toxicity of examined substances and their effect upon proline hydroxylation and its incorporation into collagenous and non-collagenous proteins The dose for stopping the movement of Tubifex tubifex worms for sodium azide moved within thelimits 8 x 1-2M to 1.5 x 1-lM Substancea Relative toxicityb Degrce of hydroxylationc Total radioactivity of imino acids in collagcnd Total activity of proline in non-collagenous proteinse 1. EDTA (disodium salt) -.5 f salicylic acid -.33 f ,4'-dipyridyl f glutathione -.75 & ,8-phenanthroline f ,7-phenanthroline & isopropyltropolon f SKF (525A)f & ,4-naphthoquinone f ,4-benzoquinone & diethyldithiocarbamate (sodium salt).23 & desferrioxamin B g > ,2'-dipyridyl & ,lO-phenanthroline f hydroxyquinoline > cuso,.5 f NaN,. f GI KCN -.51 f ,2-naphthoquinone-4-sulfonate (sodium salt) f iodoacetic acid a For studying the biological effect b, c, d 1 nidl concentration waq used. The substances are divided into three groups; 1. the group of substance which does not form a complex with Felt and does not dissociate at all or very little; 2. chelating agents forming a comparatively firm complex with Fen+; 3. substances dissociating very easily. ltelative toxicity is expressed by the logarithm of the ratio of molar concentration of the studied substance to the molar concentration of reference substance (NaNJ and refers to the dose which causcs the stopping of the movement of w-orms Tubifex tubifez. The degree of hydroxylation is expressed as the relation of [14C] hydroxyproline activity to the activity of ["C] proline in collagen protein. The total quantity of incorporated imino acids into the collagen molecule is expressed in per cent of the [W] activity of control. The quantity of Proline incorporated into thc molecule of non-collagenous proteins is expressed in per cent of the ['W] activity of control. * Refers to diethylaminoethyldiphenylpropylacetate hydrochloride (Smith ICline & French Labs. Philadelphia). g Befers to Desferalm (CIBA, Switzcrland).

7 Vol.2, S.2, 1967 M. CHVAPIL, J. HURYCH, E. EHRLICHOV~, and &I. TICHY 235 correlation exists between the extent of the inhibition of proline hydroxylation and general toxicity of the substances used. It is evidently an interference with the basic metabolic processes of the cell. These results are presented in Table 3, which includes all substances studied divided into three groups according to their properties. Group a) refers to substances with a low degree of dissociation; group b) are substances with typical chelating properties and group c) are casily dissociable substances. Correlation of relative toxicity (column 2) of the whole group of compounds with reactions (3,4 and 5) has not been found [(2 x3) r =.17, (2 x4) r =.48, (2 x5) r =.431. There exists, however, a significant correlation with substances having a low degree of dissociability [(2 x4) r =.88, (2 x5) r =.881. Proposed Concept on the Mechanism of Action of Chelating Agents and other Substances on Proline Hydroxylation and Protein Syn,thesis The selective inhibition of collagen hydroxylation may be called a specific reaction, because it is related only to chelating agents forming strong complexes with bivalent iron. This inhibition occurs promptly and within a certain range of concentrations and it is limited to proline and lysine hydroxylation only. No difference has been found in inhibition of proline or lysine hydroxylation with various chelating agents. Recently there was found [it%], that the lysine hydroxylating enzyme requires the same cofactor (ascorbate, a-ketoglutarate and Fez') as the proline hydroxylase. This favours the hypothesis that for hydroxylation of both proline and lysine there exists only one hydroxylase which is an iron-sensitive enzyme. The inhibition of incorporation of proline into collagenous and non-collagenous protein (which reflects the synthesis of these proteins) occurs at higher concentration and longer periods of incubation with chelating agents than are necessary for the inhibition of hydroxylation. The incorporation of amino acids into the collagenous molecule is more easily inhibited by chelating agents than the synthesis of globular proteins. This shows that there exist differences in biosynthetic pathways between both mentioned groups of proteins which manifest themself just at the action of chelating agents. The actual nature of this difference remains obscure. Substances without chelating properties inhibit the synthesis of proteins to the same extent (accord- ing to the concentration used), but in this case the decrease in hydroxypoline formation is secondary to the decreased incorporation of proline into a peptide which is a substrate for hydroxylation. This action is related to interferences at various metabolic steps involved in the protein synthesis, the further description of which could bc speculative only. These substances are known to interfere with oxidation-reduction processes (quinones, cytochrome c, glutathione), with transport of amino acids (glycine and other amino acids), with the metabolism of pyridoxal as an enzyme co-factor (penicillamine) etc. Owing to the non-uniform mode of action and owing to certain relation of the effect to general toxicity of this group of substances, we call this effect on proline incorporation into collagenous and non-collagenous proteins nonspecific REFERENCES 1. Hurych, J., and Chvapil, M., Biochim. Biophys. Acta, 97 (1965) Chvapil, M., EhrlichovL, E., and Hurych, J., Experientia, 22 (1966) Chvapil, M., Hurych, J., EhrlichovL, E., and Cmucha- lovii, B., Biochim. Biophys. Acta, 14 (1967) 399. Fitch, S. M., Harkness, M. L. R., and Harkness, R. D., Nature, 176 (1955) 163. Troll, W.; and Lindsley, J., J. Biol. Chem. 215 (1955) 655. Stegemann, H., 8. Physiol. Chem. 311 (1958) 41. Peterkofsky, B., and Prockop, D. J., Anal. Biochem. 4 (1962) 4. Chvapil, M., Zahradnik, R., and Cmuchalovii, B., Arch. intern. Pharmacodyn. 135 (1962) 33. Streitwieser, A., Molecular Orbital Theory for Organic Chemists, John Wiley and Sons, New Pork Hurych, J., Chvapil, M., and Nordwig, A., 2. Physiol. Chem. 348 (1967) 282. Albert, A., Selective Toxicity, Methuen, London 196. Peterkofsky, B., and Udenfriend, S., Proc. Natl. Acad. Sci. U. S. 53 (1965) 335. Hutton, Jr., J. J., Tappel, A. L., and Udenfriend, S., Arch. Biochem. Biophys. 118 (1967) 231. Kivirikko, K. J., and Prockop, D. J., Arch. Biochem. Biophys. In press. Albert, A., Federation Proc. 2 (1961) 137. Brandt, W., and Gullstrom, D. K., Symposium of Equilibrium and Rate Behaviour of Complex Ions, University of Chicago, Hurych, J., 8. Physiol. Chem. 348 (1967) 426. Hausinann, E., Federation Proc. 26 (1967) 669. M. Chvapil, J. Hurych, E. Ehrlichovii, and M. Tichgi Department of Experimental Biology Institute of Industrial Hygiene and Occupational Diseases Srobarova 48, Praha 1 - Vinohrady, Czechoslovakia