258
Biochimica et Biophysica Acta, 538 (1978) 258--267
© Elsevier/North-Holland Biomedical Press
BBA 28389 D I H Y D R O P Y R I D I N E P R E C U R S O R S OF ELASTIN CROSSLINKS
NORMAN R. DAVIS Department of Oral Biology, University of Alberta, Edmonton, T6G 2N8 (Canada)
(Received May 9th, 1977)
Summary Dihydrodesmosine and dihydroisodesmosine are dihydropyridines which are believed to be the immediate biosynthetic precursors of desmosine and isodesmosine, the stable pyridinium ion crosslinks of elastin. It has recently been reported that appreciable amounts of dihydrodesmosine and dihydroisodesmosine accumulate in elastin. In view of the ease with which such dihydropyridines are oxidized to the corresponding pyridinium ions by 02 and other mild oxidants, dihydrodesmosine and dihydroisodesmosine would not be expected to accumulate in elastic tissues. It therefore seemed appropriate to analyse elastin samples for dihydrodesmosine and dihydroisodesmosine using techniques different from these previously employed for this purpose. The results of these investigations indicate that dihydrodesmosine and dihydroisodesmosine do not accumulate to a measurable extent in elastin.
Introduction Current evidence suggests that insoluble fibers of elastin are formed by the covalent crosslinking of a polypeptide precursor, proelastin [1]. The crosslinking involves allysine (the aldehyde produced by oxidative deamination of lysine residues) as well as intact lysine residues [2--4]. A mechanism which fully accounts for the formation of desmosine and isodesmosine crosslinks has been proposed by Davis and Anwar [5]. This mechanism (summarized in Fig. 1) features the formation of desmosine (VI) and isodesmosine (IV) from dihydropyridine precursors, 1,4-dihydrodesmosine (V) and 1,2-dihydroisodesmosine (III), respectively. Model c o m p o u n d studies [5,6] have shown that aliphatic aldehyde analogues of allysine and aliphatic amine analogues of lysine react to form aldimine analogues of dehydrolysinonorleucine (A-LNL in Fig. 1) which, in turn, react with allysine analogues to give tetraalkyl-l,2-dihydropyridine analogues of dihydroisodesmosine (III, Fig. 1). Since these analogues of III are readily converted to tetra-alkyl pyridinium analogues of isodesmosine (IV) by a variety
259
R~R
/ / " r H ~ .., ~-"H RCH H
-HOH R . ~ R
-HOH > H~ .. [L RCH H
cH~v~~.d4C ' H0
RCHz / [ ~ H
~f~CHz~'L~H i
-HOH ~
RCH~"~ ": 2 ~ . ~ (A-LNL) '1...._~_.._.~
(allys;ne)
"~"~
RCH2CHO~ RINH2 RCH2CHO
H
(i
(lysine)
-HOH ~
Fig. 1. Mechanism
~:. ~ " Mr;H2/
(aldol)
CH2R
RH~H2RR •
R.._ /CHO
'"
RCH2 H H H" ~:~
.
H'~z
OHH
H CH2RR
I!
(~)
(V) I~z
of i s o d e s m o s i n e ( I V ) and d e s m o s i n e ( V I ) f o r m a t i o n in elastin. A - L N L , d e h y d r o l y s i n o -
norleueine.
of mild oxidants, the model compound studies strongly support the mechanism outlined here. Although detection of dihydrodesmosine and dihydroisodesmosine in elastin would lend further support to the mechanism proposed for elastin crosslinking, these dihydropyridines would not be expected to accumulate to the extent suggested by previous studies [7], Furthermore, any dihydrodesmosine or dihydroisodesmosine which did accumulate in vivo would not be expected to survive the conditions employed to isolate elastin [7]. Indeed, direct experience at handling a variety of tetraalkyl-l,2-dihydropyridine analogues of isodesmosine has emphasized the lability of such compounds in air, and an extensive literature survey has confirmed the great sensitivity of 1,2- 1,4- and 1,6-dihydropyridines to mild oxidizing agents [5,6,8]. In addition, the previous estimate [7] of dihydrodesmosine and dihydroisodesmosine contents was based upon techniques which have led to erroneous conclusions when applied to the study of simpler collagen crosslinks [9--11].
260 The aim of the work described in this paper was to develop specific chemical and spectrophotometric methods for measuring the quantities of dihydrodesmosine and dihydroisodesmosine which accumulate in vivo. For this purpose, samples of bovine ligamentum nuchae elastin were prepared by conventional methods and also by methods designed to minimize the spontaneous aerobic oxidation of dihydrodesmosine and dihydroisodesmosine to desmosine and isodesmosine, respectively. Materials and Methods Twice-crystallized elastase was obtained from Worthington Biochemicals. Highest purity solvents and reagents were used for the spectrophotometric and chemical studies, and Matheson research grade nitrogen (less than 1 ppm 02) was employed to deoxygenate samples and to maintain anaerobic conditions. Elastin was isolated from the ligamentum nuchae of a two-week-old calf immediately after death. In order to minimize air oxidation of any dihydrodesmosine or dihydroisodesmosine present, all buffers and reagents were purged with N2 immediately before use in order to remove 02. All experiments were performed under an atmosphere of N2 (except where otherwise indicated). Fats were extracted by boiling small, hand-cut pieces of ligament with ethanol, ethanol/ether and, finally, ether. The residue was dried in a vacuum oven at 37°C, and ground to a powder. Some samples were extracted with 0.1 M NaOH for 60 min at 98°C to remove non-elastin proteins [12]. The insoluble elastin was then washed with water until neutral, extracted with boiled ethanol, then ether, and dried in a vacuum over at 37°C. All samples were stored in sealed bottles under N2 in a N2-filled desiccator. Elastin samples were also prepared from calf and cow ligaments without protection from air, using the alkalai extraction m e t h o d of Lansing et al. [12] described above, and also using the m e t h o d of Partridge et al. [13]. Elastin samples were solubilized for spectrophotometric studies by proteolysis with elastase [14] under anaerobic conditions. Elastin (500 mg) was suspended in 50 ml 0.05 M a m m o n i u m carbonate buffer (pH 8.8) and digested with 5 mg of elastase for 24 h at 37°C under an atmosphere of N2. The soluble peptide mixture was filtered through millipore (0.45 pM), and evaporated to dryness in vacuo. The residue was then repeatedly evaporated to dryness in vacuo from aliquots of N2-purged water in order to remove traces of NH3 and CO2, and a concentrated solution of the elastase peptides in 50 ml of N2-purged water was prepared for spectrophotometric studies. l-Butyl-2-propyl-3,5-diethyl-l,2-dihydropyridine was synthesized under anaerobic conditions from a N2-purged solution of N-butylidenebutylamine and butyraldehyde, as described previously [5,6]. This dihydropyridine, which possesses a ring structure identical to that of dihydroisodesmosine, was used as a model c o m p o u n d in developing the spectrophotometric and chemical techniques used to assay elastin peptides for dihydroisodesmosine and dihydrodesmosine. All spectrophotometric experiments were conducted using a Pye Unicam SP8000 double beam recording ultraviolet spectrophotometer equipped with matched 1 cm quartz cells.
261 For amino acid analysis, elastin samples were hydrolysed for 72 h at 110°C in double-distilled, constant boiling HC1 in sealed vials under nitrogen. The hydrolysates were dried in vacuo on a rotary evaporator to remove HC1, and analysed on a Beckmann Model 120B amino acid analyser as described previously [14]. Elastin samples were suspended in water and reduced with NaBHa as described previously [ 14]. Treated and untreated elastin samples (100 mg) were stirred overnight in the dark with 8 ml of 0.1 M NaIO4 containing 40 pmol OsO4 in order to destroy any dihydrodesmosine or dihydroisodesmosine present. The effectiveness of this procedure in destroying dihydropyridines was demonstrated with the model c o m p o u n d , 1-butyl-2-propyl-3,5-diethyl-l,2-dihydropyridine. Oxidation of this c o m p o u n d was effected by treatment of an ether solution with one equivalent or less of OsO4, and stirring over 0.1 M aqueous NaIO4 under anaerobic conditions. Treatment of the 1-butyl-2-propyl-3,5-diethylpyridinium ion {produced by 02 oxidation of the model dihydropyridine) was effected in the same manner ~n order to demonstrate the stability of the pyridinium ring to OsO4/periodate. Treatment of elastin with I: was effected by stirring 100-mg samples overnight in sealed vials containing 10 ml of 0.1 M KI solution saturated with I2. Treatment with 02 was effected by stirring each 100-rag sample overnight in 10 ml of water contained in a 25-ml flask connected to an oxygen cylinder (15 lb/inch2). Results Dihydroisodesmosine is a 1,2,3,5-tetraalkyl-l,2-dihydropyridine and, as such, should gave an absorption maximum at 335 +_15 nm with an extinction coefficient in the range of 8000--13 000 in neutral or alkaline solutions [6,8]. Like other such dihydropyridines, dihydroisodesmosine should be protonated on the weakly basic ring nitrogen at low pH, yielding a species which no longer absorbs ultraviolet light above 300 nm. Because of these spectral properties, dihydroisodesmosine should be readily detected in solubilized elastin peptides, provided as little as 0.1 residue is present per 1000 residues. Such peptides should have an absorbance m a x i m u m at 335-+ 15 nm in neutral or alkaline solution. If as little as 0.1 residue of dihydroisodesmosine per 1000 is present, the absorbance at this absorbance maximum should decrease by at least 0.10 units when the pH of a 1% solution is lowered from 8 to 2. No other amino acid residues present in elastin undergo such spectral changes above 300 nm over this pH range. Therefore, spectrophotometric studies can be unambiguously interpreted. The spectrum of 1-butyl-2-propyl-3,5-diethyl-l,2-dihydropyridine, which has the same dihydropyridine ring structure as dihydroisodesmosine, is depicted in Fig. 2. Addition of HC1 to a dilute solution of this c o m p o u n d results in a large, instantaneous drop in absorbance above 300 nm as a result of ring protonation (Fig. 2b,c). If the acid solution is neutralized, the prominent absorbance maxim u m at 334 nm is immediately restored. In contrast, a neutral solution of calf elastin (94 mg in 10 ml), isolated and solubilized with elastase under anaerobic
262 A 0.6 a
A
\
1.0
0.4
0.5
c \ ,, '\
0.2
I
i
330
I
i
340 ~,(nm)
0.0 310
320
330
A[nm)
Fig. 2. S p e c t r o p h o t o m e t r i c t i t r a t i o n of 1 - b u t y l - 2 - p r o p y l - 3 , 5 - d i e t h y l - l , 2 - d i h y d r o p y r i d i n e in d e o x y g e n a t e d m e t h a n o l : a, u n t r e a t e d s o l u t i o n ; b , t r e a t e d w i t h 0.3 3 m e q u i v , of 12 M HC1; c, t r e a t e d w i t h ~>1 m e q u i v , of 12 M HC1. P r o m p t a d d i t i o n of s t r o n g alkali r e s t o r e s t h e s p e c t r u m s h o w n in (a). Fig. 3. S p e c t r o s p h o t o m e t r i c t i t r a t i o n of elastase-solubliized elastin p e p t i d e s (9.3 m g . ml-1 ). p H : a, 7.0; b, 2.0; c, 11.0; d, 1 1 . 0 , a f t e r N a B H 4 r e d u c t i o n .
conditions, exhibits no absorbance m a x i m u m above 300 nm and, m ore importantly, exhibits no measureable loss of absorbance above 300 nm when the pH is lowered to 2 with 12 M HC1 (Fig. 3a,b). F u r t h e r m o r e , highly concent rat ed solutions (containing 94 mg protein/ml) exhibit no absorbance m a x i m u m above 300 nm and exhibit no loss of absorbance above 300 nm upon acidification with 12 M HC1. R e d u c t i o n of elastin peptides with NaBH4 (destroying desmosine and isodesmosine) lowers the absorbance at 325 nm (pH 11) by 75% (Fig. 3c,d). Hence, even the desmosines, which absorb an order of magnitude more intensely at 278 nm than at 325 nm, show detectable spectral differences at 325 nm when their c h r o m o p h o r i c structure is altered. Clearly, dihydroisodesmosine, which should absorb at least an order of magnitude more strongly at 325 nm than do the desmosines, c a nnot be present in significant a m o u n t s in calf elastin in view of the absence of an absorbance m a x i m u m at 335 ± 15 nm in neutral solution and the absence of significant spectral changes upon acidification. Even 0.02 residue per 1000 of dihydroisodesmosine should give a detectable absorbance difference between pH 2 and 8 (about 0.01 absorbance unit difference). Such a difference is not observed. T he results o f the s p e c t r o p h o t o m e t r i c experiments are supported by the results o f specific chemical degradation experiments which are discussed below. These experiments are based upon the susceptibility of 1,2-dihydropyridines and 1,4-dihydropyridines, such as dihydroisodesmosine and dihydrodesmosine, respectively, to oxidative cleavage by periodate solutions containing catalytic amounts o f OsO4 (Fig. 4). This mild oxidative procedure involves addition of OsO4 to the double bond, yielding an osmate ester which is t hen cleaved by periodate, regenerating the OsO4 [15]. Addition of progressively larger a m o u n ts of OsO4 to 1-butyl-2-propyl-3,5-diethyl-l,2-dihydropyridine in ether cases a progressive, irreversible loss of absorbance at the characteristic absorbance m a x i m u m as the d i h y d r o p y r i d i n e is converted to the osmate ester. When a solution (A 1.6) of this d i h y d r o p y r i d i n e containing less than one equivalent o f OsO4 is shaken with 0.1 M NaIO4 in water, the absorbance at 334 nm rapidly falls to zero as a result of ring cleavage by periodate and catalytic amounts of
263 (P~O 2
Io2
Or III
0s04
Biochimica et Biophysica Acta, 538 (1978) 258--267
© Elsevier/North-Holland Biomedical Press
BBA 28389 D I H Y D R O P Y R I D I N E P R E C U R S O R S OF ELASTIN CROSSLINKS
NORMAN R. DAVIS Department of Oral Biology, University of Alberta, Edmonton, T6G 2N8 (Canada)
(Received May 9th, 1977)
Summary Dihydrodesmosine and dihydroisodesmosine are dihydropyridines which are believed to be the immediate biosynthetic precursors of desmosine and isodesmosine, the stable pyridinium ion crosslinks of elastin. It has recently been reported that appreciable amounts of dihydrodesmosine and dihydroisodesmosine accumulate in elastin. In view of the ease with which such dihydropyridines are oxidized to the corresponding pyridinium ions by 02 and other mild oxidants, dihydrodesmosine and dihydroisodesmosine would not be expected to accumulate in elastic tissues. It therefore seemed appropriate to analyse elastin samples for dihydrodesmosine and dihydroisodesmosine using techniques different from these previously employed for this purpose. The results of these investigations indicate that dihydrodesmosine and dihydroisodesmosine do not accumulate to a measurable extent in elastin.
Introduction Current evidence suggests that insoluble fibers of elastin are formed by the covalent crosslinking of a polypeptide precursor, proelastin [1]. The crosslinking involves allysine (the aldehyde produced by oxidative deamination of lysine residues) as well as intact lysine residues [2--4]. A mechanism which fully accounts for the formation of desmosine and isodesmosine crosslinks has been proposed by Davis and Anwar [5]. This mechanism (summarized in Fig. 1) features the formation of desmosine (VI) and isodesmosine (IV) from dihydropyridine precursors, 1,4-dihydrodesmosine (V) and 1,2-dihydroisodesmosine (III), respectively. Model c o m p o u n d studies [5,6] have shown that aliphatic aldehyde analogues of allysine and aliphatic amine analogues of lysine react to form aldimine analogues of dehydrolysinonorleucine (A-LNL in Fig. 1) which, in turn, react with allysine analogues to give tetraalkyl-l,2-dihydropyridine analogues of dihydroisodesmosine (III, Fig. 1). Since these analogues of III are readily converted to tetra-alkyl pyridinium analogues of isodesmosine (IV) by a variety
259
R~R
/ / " r H ~ .., ~-"H RCH H
-HOH R . ~ R
-HOH > H~ .. [L RCH H
cH~v~~.d4C ' H0
RCHz / [ ~ H
~f~CHz~'L~H i
-HOH ~
RCH~"~ ": 2 ~ . ~ (A-LNL) '1...._~_.._.~
(allys;ne)
"~"~
RCH2CHO~ RINH2 RCH2CHO
H
(i
(lysine)
-HOH ~
Fig. 1. Mechanism
~:. ~ " Mr;H2/
(aldol)
CH2R
RH~H2RR •
R.._ /CHO
'"
RCH2 H H H" ~:~
.
H'~z
OHH
H CH2RR
I!
(~)
(V) I~z
of i s o d e s m o s i n e ( I V ) and d e s m o s i n e ( V I ) f o r m a t i o n in elastin. A - L N L , d e h y d r o l y s i n o -
norleueine.
of mild oxidants, the model compound studies strongly support the mechanism outlined here. Although detection of dihydrodesmosine and dihydroisodesmosine in elastin would lend further support to the mechanism proposed for elastin crosslinking, these dihydropyridines would not be expected to accumulate to the extent suggested by previous studies [7], Furthermore, any dihydrodesmosine or dihydroisodesmosine which did accumulate in vivo would not be expected to survive the conditions employed to isolate elastin [7]. Indeed, direct experience at handling a variety of tetraalkyl-l,2-dihydropyridine analogues of isodesmosine has emphasized the lability of such compounds in air, and an extensive literature survey has confirmed the great sensitivity of 1,2- 1,4- and 1,6-dihydropyridines to mild oxidizing agents [5,6,8]. In addition, the previous estimate [7] of dihydrodesmosine and dihydroisodesmosine contents was based upon techniques which have led to erroneous conclusions when applied to the study of simpler collagen crosslinks [9--11].
260 The aim of the work described in this paper was to develop specific chemical and spectrophotometric methods for measuring the quantities of dihydrodesmosine and dihydroisodesmosine which accumulate in vivo. For this purpose, samples of bovine ligamentum nuchae elastin were prepared by conventional methods and also by methods designed to minimize the spontaneous aerobic oxidation of dihydrodesmosine and dihydroisodesmosine to desmosine and isodesmosine, respectively. Materials and Methods Twice-crystallized elastase was obtained from Worthington Biochemicals. Highest purity solvents and reagents were used for the spectrophotometric and chemical studies, and Matheson research grade nitrogen (less than 1 ppm 02) was employed to deoxygenate samples and to maintain anaerobic conditions. Elastin was isolated from the ligamentum nuchae of a two-week-old calf immediately after death. In order to minimize air oxidation of any dihydrodesmosine or dihydroisodesmosine present, all buffers and reagents were purged with N2 immediately before use in order to remove 02. All experiments were performed under an atmosphere of N2 (except where otherwise indicated). Fats were extracted by boiling small, hand-cut pieces of ligament with ethanol, ethanol/ether and, finally, ether. The residue was dried in a vacuum oven at 37°C, and ground to a powder. Some samples were extracted with 0.1 M NaOH for 60 min at 98°C to remove non-elastin proteins [12]. The insoluble elastin was then washed with water until neutral, extracted with boiled ethanol, then ether, and dried in a vacuum over at 37°C. All samples were stored in sealed bottles under N2 in a N2-filled desiccator. Elastin samples were also prepared from calf and cow ligaments without protection from air, using the alkalai extraction m e t h o d of Lansing et al. [12] described above, and also using the m e t h o d of Partridge et al. [13]. Elastin samples were solubilized for spectrophotometric studies by proteolysis with elastase [14] under anaerobic conditions. Elastin (500 mg) was suspended in 50 ml 0.05 M a m m o n i u m carbonate buffer (pH 8.8) and digested with 5 mg of elastase for 24 h at 37°C under an atmosphere of N2. The soluble peptide mixture was filtered through millipore (0.45 pM), and evaporated to dryness in vacuo. The residue was then repeatedly evaporated to dryness in vacuo from aliquots of N2-purged water in order to remove traces of NH3 and CO2, and a concentrated solution of the elastase peptides in 50 ml of N2-purged water was prepared for spectrophotometric studies. l-Butyl-2-propyl-3,5-diethyl-l,2-dihydropyridine was synthesized under anaerobic conditions from a N2-purged solution of N-butylidenebutylamine and butyraldehyde, as described previously [5,6]. This dihydropyridine, which possesses a ring structure identical to that of dihydroisodesmosine, was used as a model c o m p o u n d in developing the spectrophotometric and chemical techniques used to assay elastin peptides for dihydroisodesmosine and dihydrodesmosine. All spectrophotometric experiments were conducted using a Pye Unicam SP8000 double beam recording ultraviolet spectrophotometer equipped with matched 1 cm quartz cells.
261 For amino acid analysis, elastin samples were hydrolysed for 72 h at 110°C in double-distilled, constant boiling HC1 in sealed vials under nitrogen. The hydrolysates were dried in vacuo on a rotary evaporator to remove HC1, and analysed on a Beckmann Model 120B amino acid analyser as described previously [14]. Elastin samples were suspended in water and reduced with NaBHa as described previously [ 14]. Treated and untreated elastin samples (100 mg) were stirred overnight in the dark with 8 ml of 0.1 M NaIO4 containing 40 pmol OsO4 in order to destroy any dihydrodesmosine or dihydroisodesmosine present. The effectiveness of this procedure in destroying dihydropyridines was demonstrated with the model c o m p o u n d , 1-butyl-2-propyl-3,5-diethyl-l,2-dihydropyridine. Oxidation of this c o m p o u n d was effected by treatment of an ether solution with one equivalent or less of OsO4, and stirring over 0.1 M aqueous NaIO4 under anaerobic conditions. Treatment of the 1-butyl-2-propyl-3,5-diethylpyridinium ion {produced by 02 oxidation of the model dihydropyridine) was effected in the same manner ~n order to demonstrate the stability of the pyridinium ring to OsO4/periodate. Treatment of elastin with I: was effected by stirring 100-mg samples overnight in sealed vials containing 10 ml of 0.1 M KI solution saturated with I2. Treatment with 02 was effected by stirring each 100-rag sample overnight in 10 ml of water contained in a 25-ml flask connected to an oxygen cylinder (15 lb/inch2). Results Dihydroisodesmosine is a 1,2,3,5-tetraalkyl-l,2-dihydropyridine and, as such, should gave an absorption maximum at 335 +_15 nm with an extinction coefficient in the range of 8000--13 000 in neutral or alkaline solutions [6,8]. Like other such dihydropyridines, dihydroisodesmosine should be protonated on the weakly basic ring nitrogen at low pH, yielding a species which no longer absorbs ultraviolet light above 300 nm. Because of these spectral properties, dihydroisodesmosine should be readily detected in solubilized elastin peptides, provided as little as 0.1 residue is present per 1000 residues. Such peptides should have an absorbance m a x i m u m at 335-+ 15 nm in neutral or alkaline solution. If as little as 0.1 residue of dihydroisodesmosine per 1000 is present, the absorbance at this absorbance maximum should decrease by at least 0.10 units when the pH of a 1% solution is lowered from 8 to 2. No other amino acid residues present in elastin undergo such spectral changes above 300 nm over this pH range. Therefore, spectrophotometric studies can be unambiguously interpreted. The spectrum of 1-butyl-2-propyl-3,5-diethyl-l,2-dihydropyridine, which has the same dihydropyridine ring structure as dihydroisodesmosine, is depicted in Fig. 2. Addition of HC1 to a dilute solution of this c o m p o u n d results in a large, instantaneous drop in absorbance above 300 nm as a result of ring protonation (Fig. 2b,c). If the acid solution is neutralized, the prominent absorbance maxim u m at 334 nm is immediately restored. In contrast, a neutral solution of calf elastin (94 mg in 10 ml), isolated and solubilized with elastase under anaerobic
262 A 0.6 a
A
\
1.0
0.4
0.5
c \ ,, '\
0.2
I
i
330
I
i
340 ~,(nm)
0.0 310
320
330
A[nm)
Fig. 2. S p e c t r o p h o t o m e t r i c t i t r a t i o n of 1 - b u t y l - 2 - p r o p y l - 3 , 5 - d i e t h y l - l , 2 - d i h y d r o p y r i d i n e in d e o x y g e n a t e d m e t h a n o l : a, u n t r e a t e d s o l u t i o n ; b , t r e a t e d w i t h 0.3 3 m e q u i v , of 12 M HC1; c, t r e a t e d w i t h ~>1 m e q u i v , of 12 M HC1. P r o m p t a d d i t i o n of s t r o n g alkali r e s t o r e s t h e s p e c t r u m s h o w n in (a). Fig. 3. S p e c t r o s p h o t o m e t r i c t i t r a t i o n of elastase-solubliized elastin p e p t i d e s (9.3 m g . ml-1 ). p H : a, 7.0; b, 2.0; c, 11.0; d, 1 1 . 0 , a f t e r N a B H 4 r e d u c t i o n .
conditions, exhibits no absorbance m a x i m u m above 300 nm and, m ore importantly, exhibits no measureable loss of absorbance above 300 nm when the pH is lowered to 2 with 12 M HC1 (Fig. 3a,b). F u r t h e r m o r e , highly concent rat ed solutions (containing 94 mg protein/ml) exhibit no absorbance m a x i m u m above 300 nm and exhibit no loss of absorbance above 300 nm upon acidification with 12 M HC1. R e d u c t i o n of elastin peptides with NaBH4 (destroying desmosine and isodesmosine) lowers the absorbance at 325 nm (pH 11) by 75% (Fig. 3c,d). Hence, even the desmosines, which absorb an order of magnitude more intensely at 278 nm than at 325 nm, show detectable spectral differences at 325 nm when their c h r o m o p h o r i c structure is altered. Clearly, dihydroisodesmosine, which should absorb at least an order of magnitude more strongly at 325 nm than do the desmosines, c a nnot be present in significant a m o u n t s in calf elastin in view of the absence of an absorbance m a x i m u m at 335 ± 15 nm in neutral solution and the absence of significant spectral changes upon acidification. Even 0.02 residue per 1000 of dihydroisodesmosine should give a detectable absorbance difference between pH 2 and 8 (about 0.01 absorbance unit difference). Such a difference is not observed. T he results o f the s p e c t r o p h o t o m e t r i c experiments are supported by the results o f specific chemical degradation experiments which are discussed below. These experiments are based upon the susceptibility of 1,2-dihydropyridines and 1,4-dihydropyridines, such as dihydroisodesmosine and dihydrodesmosine, respectively, to oxidative cleavage by periodate solutions containing catalytic amounts o f OsO4 (Fig. 4). This mild oxidative procedure involves addition of OsO4 to the double bond, yielding an osmate ester which is t hen cleaved by periodate, regenerating the OsO4 [15]. Addition of progressively larger a m o u n ts of OsO4 to 1-butyl-2-propyl-3,5-diethyl-l,2-dihydropyridine in ether cases a progressive, irreversible loss of absorbance at the characteristic absorbance m a x i m u m as the d i h y d r o p y r i d i n e is converted to the osmate ester. When a solution (A 1.6) of this d i h y d r o p y r i d i n e containing less than one equivalent o f OsO4 is shaken with 0.1 M NaIO4 in water, the absorbance at 334 nm rapidly falls to zero as a result of ring cleavage by periodate and catalytic amounts of
263 (P~O 2
Io2
Or III
0s04
