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Analysis of the ultrastructure of vitrosin fibers on the basis of the polarization optical method
326
BIOCHIMICA ET BIOPHYSICA ACTA
ANALYSIS OF THE ULTRASTRUCTURE
VOL. 1 1
(1953)
OF VITROSIN FIBERS
ON T H E B A S I S O F T H E P O L A R I Z A T I O N
OPTICAL METHOD
by A. G E D E O N
MATOLTSY
Department o/Dermatology, Harvard Medical School, and Dermatological Laboratories, Massachusetts General Hospital, Boston ~4, Massachusetts ( U.S.M .)
A previous paper described the isolation and properties of a viscous thixotropic fibrous protein, named vitrosin, of the vitreous body of the eye 4. In the present study fibers were prepared from vitrosin, and their form and crystalline double refraction were investigated; from these data conclusions were drawn about the shape and molecular structure of the submicroscopic particles of vitrosin.
Preparation o/ vitrosin fibers Vitrosin w a s p r e p a r e d f r o m t h e v i t r e o u s b o d y of 50 fresh cattle eyes according to a n l e t h o d described in a p r e v i o u s p a p e r 4. T h e p r e c i p i t a t e d v i t r o s i n w a s dialyzed for 48 h o u r s a g a i n s t distilled w a t e r a t 5 ° C. A f t e r this, fibers were pulled f r o m t h e s t i c k y precipitate w i t h t h e help of glass hooks. T h e fibers were placed on a celluloid grid a n d dried in a desiccator at r o o m t e m p e r a t u r e . T e n fibers were selected, e a c h of w h i c h s h o w e d i n t e n s e u n i a x i a l double refraction a n d m e a s u r e d 8 o - 1 2 o / z in d i a m e t e r . T h e s e fibers were u s e d in t h e p r e s e n t s t u d y .
Determination o~/orm and crystalline double re/faction o/ vitrosin fibers Both form and crystalline double refraction of vitrosin fibers were determined according to AMBRONN'S immersion technique 1. The fibers were immersed in various solutions of different refractive indices, viz.: water: r.33, ethanol: 1.36, chloroform: 1.44, o-xylene : 1.5o, and aniline : 1.58. After a 24-hour immersion, the double refraction was determined with a Leitz polarization microscope. The retardation (A A) was measured with the Berek compensator in monochromatic light of 540 m/, wave length. Thickness (d) was measured by focusing the microscope first on the top, then on the bottom of the fiber, and reading the difference on the micrometer scale. The double refraction (nji -- n±) was calculated from the equation : AA (n H- - n±) -
d
The average values of the double refraction in the various solutions were plotted as ordinates against the respective refractive indices of the solutions (%). A hyperbolic curve was obtained, as shown in Fig. I. The value of crystalline double refraction of the fiber was derived from the minimum of the curve at about n 2 = 1.5o. At this point the form double refraction is eliminated because the refractive index of the submicroscopic particles is equal to that of the Re/erences p. 328.
VOL. 1 1
(~953)
ULTRASTRUCTURE OF VITROSIN FIBERS
327
immersion fluid. The residual double refraction is then ÷3o equal to the crystalline double refraction, which is ,,,-nz 14" lO -4 (Fig. I). ~10-, By difference, the form double refraction in water is 9" IO-4.
Interpretatio~ o/the results
÷20
The fact that vitrosin fibers show crystalline as well as form double refraction indicates that their submicroscopic particles must have considerable regularity of structure and orientation. According to .~ WIENER'S theory ~, the positive form double refraction indicates that the submicroscopic particles are oriented parallel to the fiber's longitudinal axis and have a rodlet form. The rodlet shape of the submicroscopic • - - - 4 - particles is compatible with the electron microscopic investigation of vitrosin, which showed that vitrosin is o made up of long, thin filaments, measuring 2o0-300 A in width 3, 4. - ~.3 t.4 13 1.6 The positive crystalline double refraction indin2~ cates that vitrosin particles have a crystalline molecular structure, oriented parallel to their longi- Fig. I. R o d l e t b i r e f r i n g e n c e c u r v e of v i t r o s i n fibers. A b s c i s s a : d o u b l e tudinal axis. In fibrous proteins such a molecular re fra c t i on. O r d i n a t e : r e f r a c t i v e structure usually corresponds to oriented polypeptide i n d e x of i m m e r s i o n fluids. chains 5. In biological objects the value of form double refraction is known to be always smaller than that of the crystalline double refraction 2. The double refraction of vitrosin fibers, observed here, conforms to this rule.
SUMMARY I. F i b e r s were p r e p a r e d from a s t r u c t u r a l p r o t e i n (vitrosin) of t h e v i t r e o u s b o d y a n d t h e i r form a n d c r y s t a l l i n e d o u b l e r e f r a c t i o n were d e t e r m i n e d . 2. T h e form d o u b l e r e f r a c t i o n was c a l c u l a t e d as 9" lO-4, t h e c r y s t a l l i n e d o u b l e r e f r a c t i o n as 14 • lO -4. 3. On t h e b a s i s of t h e s e d a t a , a n d WIENER'S t h e o r y , t h e c onc l us i on w a s d r a w n t h a t v i t r o s i n fibers are c o m p o s e d of r o d l e t - s h a p e d s u b m i c r o s c o p i c partic l e s , w h i c h h a v e a l o n g i t u d i n a l l y o r i e n t e d molecular structure.
Rt~SUMt~ i. No us a v o n s p r6par6 des fibres A p a r t i r d ' u n e p r o t 6 i n e s t r u c t u r a l e (vitrosine) du c orps v i t r 6 e t nou s a v o n s d ~ t e r m i n 6 la d o u b l e r 6 f r a c t i o n de forme et l a d o u b l e r ~ f r a c t i o n c ri s t a l l i ne . 2. P o u r la d o u b l e r 6 f r a c t i o n de forme, le c a l c u l n o u s a donn6 u n e v a l e u r de 9" xo-4, p o u r l a double r 6 f r a c t i o n cristaUine, 14 • lO -4. 3. E n n ous b a s a n t sur ces donn6es et s u r la th6orie de WIENER, nOUS c o n c l u o n s q u e les fibres de v i t r o s i n e se c o m p o s e n t de p a r t i c u l e s s u b m i c r o s c o p i q u e s ~ forme de bAtonnets, a y a n t u n e s t r u c t u r e mol~culaire ~ o r i e n t a t i o n l o n g i t u d i n a l e .
Re/erences p. 328.
328
a. GEDEON MATOLTSY
VOL. 11 (1953)
Z USAMMENFASSUNG I. Aus einem Strukturprotein (Vitrosin) des GlaskOrpers wurden Fasern dargestellt und ihre Form- und Eigendoppelbrechung bestimmt. 2. Die Formdoppclbrechung wurde zu 9' lO-4, die Eigendoppelbrechung zu 14. lO-4 berechnet. 3. Auf grund dieser Daten und ~vVlENER'STheorie wurde tier Schluss gezogen, dass die Vitrosinfasern aus stAbchenf6rmigen, submikroskopischen Teilchen zusammengesetzt sind, die eine longitudinal orientierte Molekularstruktur haben. REFERENCES 1 AMBRONN-FREV, Das Polarizationsmikroskop, Leipzig. 1926. 2 A. FREY-W~CsSLING, Submicroscopic Morphology, Elsevier Publishing Company. New York. 1948. p. 62. a A. G. MATOLTSY, J. GRoss, AND A. GRIGNOLO, Proc. Soc. Exp. Biol..IVied., 76 (1951) 857. 4 A. G. MATOLTS'¢,J. Gen. Physiol., 36 (I952) 29. s D. F. WAUGH, Ann. N . Y . Acad. Sci., 50 (1951) 835. e O. WIENER, Abh. sachs. Ges. Wiss., 33 (1912) 507. R e c e i v e d F e b r u a r y I 8 t h , 1953
BIOCHIMICA ET BIOPHYSICA ACTA
ANALYSIS OF THE ULTRASTRUCTURE
VOL. 1 1
(1953)
OF VITROSIN FIBERS
ON T H E B A S I S O F T H E P O L A R I Z A T I O N
OPTICAL METHOD
by A. G E D E O N
MATOLTSY
Department o/Dermatology, Harvard Medical School, and Dermatological Laboratories, Massachusetts General Hospital, Boston ~4, Massachusetts ( U.S.M .)
A previous paper described the isolation and properties of a viscous thixotropic fibrous protein, named vitrosin, of the vitreous body of the eye 4. In the present study fibers were prepared from vitrosin, and their form and crystalline double refraction were investigated; from these data conclusions were drawn about the shape and molecular structure of the submicroscopic particles of vitrosin.
Preparation o/ vitrosin fibers Vitrosin w a s p r e p a r e d f r o m t h e v i t r e o u s b o d y of 50 fresh cattle eyes according to a n l e t h o d described in a p r e v i o u s p a p e r 4. T h e p r e c i p i t a t e d v i t r o s i n w a s dialyzed for 48 h o u r s a g a i n s t distilled w a t e r a t 5 ° C. A f t e r this, fibers were pulled f r o m t h e s t i c k y precipitate w i t h t h e help of glass hooks. T h e fibers were placed on a celluloid grid a n d dried in a desiccator at r o o m t e m p e r a t u r e . T e n fibers were selected, e a c h of w h i c h s h o w e d i n t e n s e u n i a x i a l double refraction a n d m e a s u r e d 8 o - 1 2 o / z in d i a m e t e r . T h e s e fibers were u s e d in t h e p r e s e n t s t u d y .
Determination o~/orm and crystalline double re/faction o/ vitrosin fibers Both form and crystalline double refraction of vitrosin fibers were determined according to AMBRONN'S immersion technique 1. The fibers were immersed in various solutions of different refractive indices, viz.: water: r.33, ethanol: 1.36, chloroform: 1.44, o-xylene : 1.5o, and aniline : 1.58. After a 24-hour immersion, the double refraction was determined with a Leitz polarization microscope. The retardation (A A) was measured with the Berek compensator in monochromatic light of 540 m/, wave length. Thickness (d) was measured by focusing the microscope first on the top, then on the bottom of the fiber, and reading the difference on the micrometer scale. The double refraction (nji -- n±) was calculated from the equation : AA (n H- - n±) -
d
The average values of the double refraction in the various solutions were plotted as ordinates against the respective refractive indices of the solutions (%). A hyperbolic curve was obtained, as shown in Fig. I. The value of crystalline double refraction of the fiber was derived from the minimum of the curve at about n 2 = 1.5o. At this point the form double refraction is eliminated because the refractive index of the submicroscopic particles is equal to that of the Re/erences p. 328.
VOL. 1 1
(~953)
ULTRASTRUCTURE OF VITROSIN FIBERS
327
immersion fluid. The residual double refraction is then ÷3o equal to the crystalline double refraction, which is ,,,-nz 14" lO -4 (Fig. I). ~10-, By difference, the form double refraction in water is 9" IO-4.
Interpretatio~ o/the results
÷20
The fact that vitrosin fibers show crystalline as well as form double refraction indicates that their submicroscopic particles must have considerable regularity of structure and orientation. According to .~ WIENER'S theory ~, the positive form double refraction indicates that the submicroscopic particles are oriented parallel to the fiber's longitudinal axis and have a rodlet form. The rodlet shape of the submicroscopic • - - - 4 - particles is compatible with the electron microscopic investigation of vitrosin, which showed that vitrosin is o made up of long, thin filaments, measuring 2o0-300 A in width 3, 4. - ~.3 t.4 13 1.6 The positive crystalline double refraction indin2~ cates that vitrosin particles have a crystalline molecular structure, oriented parallel to their longi- Fig. I. R o d l e t b i r e f r i n g e n c e c u r v e of v i t r o s i n fibers. A b s c i s s a : d o u b l e tudinal axis. In fibrous proteins such a molecular re fra c t i on. O r d i n a t e : r e f r a c t i v e structure usually corresponds to oriented polypeptide i n d e x of i m m e r s i o n fluids. chains 5. In biological objects the value of form double refraction is known to be always smaller than that of the crystalline double refraction 2. The double refraction of vitrosin fibers, observed here, conforms to this rule.
SUMMARY I. F i b e r s were p r e p a r e d from a s t r u c t u r a l p r o t e i n (vitrosin) of t h e v i t r e o u s b o d y a n d t h e i r form a n d c r y s t a l l i n e d o u b l e r e f r a c t i o n were d e t e r m i n e d . 2. T h e form d o u b l e r e f r a c t i o n was c a l c u l a t e d as 9" lO-4, t h e c r y s t a l l i n e d o u b l e r e f r a c t i o n as 14 • lO -4. 3. On t h e b a s i s of t h e s e d a t a , a n d WIENER'S t h e o r y , t h e c onc l us i on w a s d r a w n t h a t v i t r o s i n fibers are c o m p o s e d of r o d l e t - s h a p e d s u b m i c r o s c o p i c partic l e s , w h i c h h a v e a l o n g i t u d i n a l l y o r i e n t e d molecular structure.
Rt~SUMt~ i. No us a v o n s p r6par6 des fibres A p a r t i r d ' u n e p r o t 6 i n e s t r u c t u r a l e (vitrosine) du c orps v i t r 6 e t nou s a v o n s d ~ t e r m i n 6 la d o u b l e r 6 f r a c t i o n de forme et l a d o u b l e r ~ f r a c t i o n c ri s t a l l i ne . 2. P o u r la d o u b l e r 6 f r a c t i o n de forme, le c a l c u l n o u s a donn6 u n e v a l e u r de 9" xo-4, p o u r l a double r 6 f r a c t i o n cristaUine, 14 • lO -4. 3. E n n ous b a s a n t sur ces donn6es et s u r la th6orie de WIENER, nOUS c o n c l u o n s q u e les fibres de v i t r o s i n e se c o m p o s e n t de p a r t i c u l e s s u b m i c r o s c o p i q u e s ~ forme de bAtonnets, a y a n t u n e s t r u c t u r e mol~culaire ~ o r i e n t a t i o n l o n g i t u d i n a l e .
Re/erences p. 328.
328
a. GEDEON MATOLTSY
VOL. 11 (1953)
Z USAMMENFASSUNG I. Aus einem Strukturprotein (Vitrosin) des GlaskOrpers wurden Fasern dargestellt und ihre Form- und Eigendoppelbrechung bestimmt. 2. Die Formdoppclbrechung wurde zu 9' lO-4, die Eigendoppelbrechung zu 14. lO-4 berechnet. 3. Auf grund dieser Daten und ~vVlENER'STheorie wurde tier Schluss gezogen, dass die Vitrosinfasern aus stAbchenf6rmigen, submikroskopischen Teilchen zusammengesetzt sind, die eine longitudinal orientierte Molekularstruktur haben. REFERENCES 1 AMBRONN-FREV, Das Polarizationsmikroskop, Leipzig. 1926. 2 A. FREY-W~CsSLING, Submicroscopic Morphology, Elsevier Publishing Company. New York. 1948. p. 62. a A. G. MATOLTSY, J. GRoss, AND A. GRIGNOLO, Proc. Soc. Exp. Biol..IVied., 76 (1951) 857. 4 A. G. MATOLTS'¢,J. Gen. Physiol., 36 (I952) 29. s D. F. WAUGH, Ann. N . Y . Acad. Sci., 50 (1951) 835. e O. WIENER, Abh. sachs. Ges. Wiss., 33 (1912) 507. R e c e i v e d F e b r u a r y I 8 t h , 1953