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Penetration rate of dimethyladipimidate. Use of gelatin gels as a model system
Micron and Microocopica Acta, Vol. 19, No. 3. pp. 137-139, 1988. Printed In Great Britain.
0739—6260.88 83.00+1)0(1 Pergamon Press plc
PENETRATION RATE OF DIMETHYLADIPIMIDATE. USE OF GELATIN GELS AS A MODEL SYSTEM MARGARET TZAPHLIDOU*t and DEMETRIOS
P.
MATTHOPOULOSt
Laboratories of *Medical Physics and tGeneral Biology, Medical School, University of loannina, P.O. Box 1186, loannina 45110, Greece (Received 11 March 1988)
Abstract—The rate of dimethyladipimidate penetration Into gelatin gels is influenced primarily by the fixative concentration, pH, temperature and type of buffer. Buffer penetration into the same gels is more rapid than dimethyladipimidate penetration. Index key words: Penetration, dimethyladipimidate, gelatin gels.
INTRODUCTION The bifunctional cross-linking reagent (fixative) dimethyladipimidate (DMA) belongs to the class of diimidoesters and it has been employed to cross-link a variety of proteins (Anderson and Fisher, 1981; Pennathur-Das et al., 1984) and cellular membranes (Ji, 1974; Henriques and Park, 1978). It has also been used in electron and light microscopy to study rat liver (Hassell and Hand, 1974), collagen (Tzaphlidou, 1987) and monkey kidney CV! and COS1 cells (Matthopoulos and Tzaphlidou, 1987, 1988). Even though diimidoesters and particularly dimethyladipimidate have been applied to various biological materials, their penetration rate has not yet been studied. The penetration rate of a fixative is of paramount importance as the depth of penetration is the same as the depth of fixation. Fixative penetration can be calculated by the expression d=k\/t (Medawar, 1941), where d=depth of fixative penetration in mm, = time in mm and k = constant (coefficient of diffusibility of the fixative). The primary purpose underlying the work carried out in the present investigation has been to account for the factors determining the penetration rate of dimethyladipimidate into gelatin gels.
MATERIALS AND METHODS Gelatin solutions were used by Davis and Tabor (1963) and Coetzee and van der Merwe (1985) as a model system to study the penetration rate and the cross-linking effect of aldehydes on a protein. This system allows accurately defined material to be used for analysis and was therefore employed here. The dimethyladipimidate dihydrochloride (DMA) was from Sigma Chemical Co. Fixative solutions were prepared immediately prior to use as described previously (Tzaphlidou, 1987; Tzaphlidou and Chapman, 1984). The buffer types used were: (i) Tris, (ii) Tris Dulbecco’s phosphate buffer saline (PBS) and (iii) Tris Earle’s basal salt solution (BSS). The penetration of DMA and buffer into gelatin was estimated as described by Coetzee and van der Merwe (1985) with minor modifications. Glass tubes (8 mm i.d., 30mm long) were closed at one end with Paraflim and filled with a 10% bacteriological grade aqueous gelatin solution. After cooling, the Parafilm was removed and the tubes were stood in 5 ml fixative solutions in 25 ml glass beakers. After the required fixation time, the cross-linked gelatin was removed by immersing the tubes in hot water at 60°Cfor 2 mm. During this time, the uncrosslinked gelatin is melted and can be poured out. Rinsing of the tubes with water at the same
~ Author to whom correspondence should be sent. 137
I 38
Mirgarcl l 7aphlidou and Demetrto~P. MatthopouIo~
temperature removes the last traces of uncrosslinked gelatin while the cross-linked gelatin forms an insoluble plug in the end of the tube. The length of this plug was measured to the nearest 0.1 mm. The buffer penetration into gelatin was monitored by the change of the indicator color added to the gel. For this purpose, a few drops of a 0.5% aqueous solution of phenol red were added to the gelatin solution before filling the tubes. All values given are the mean of at least four separate repetitions. RESULTS Dimethyladipimidate penetrates into gelatin gel at a rate of 90 ~tm min ‘for the first 10 mm and then drops off (Table I). The penetration rate of DMA into the gel is 21 ~tm min~ for fixation time 2~h which is the time chosen for tissue cross-linking (Tzaphlidou, 1987). The nature of the fixed material plays a role in penetration. The penetration by this diimidoester into 10% gelatin gel that contains O.5% bovine serum albumin is 150 ~immm ‘ in -
.
.
.
.
Table I. Penetration of I ~ mg, nil dimethyladipimidate in 100 mM Tris pH 9.5 into 0% gelatin gel
10 mm. declining to 18 ~tm min in 2 h. When the gelatin is dissolved in Dulbecco’s phosphate buffer saline (pH 7.4) instead of water, the rate of DMA penetration into the gel is 110 ~tm min ‘if thedepthofpenetrationafter 10 ministakeninto consideration. If the penetration rate is calculated for 2 h after contact of the gel with the fixative, the penetration rate is approximately IS j.tm mm In both cases, the values for rates after 2 h are comparable to that obtained for aqueous solution of gelatin (Table I). If, however, the rates after tO mm are taken into consideration, they are high compared with that of Table I. The rate of penetration, and therefore k, depends also on the concentration of DMA (Table 2). An increased concentration of DMA from I mg/mI to 15 mg/mI enhances its rate of penetration while concentrations greater than 15 mg/mI result in a decrease in penetration rate. The value of k is markedly influenced by the pH of the fixative (Table 3). DMA penetrates fasteratroomtemperaturethaninthecold.After 2 h fixation at room temperature, the value of k obtained is 0.28 while at 7C it is 0.24. Penetration is only slightly affected by the molarity of the buffer (Table 4). The effect is -
Table 3. Penetration of IS mg, ml dimethyladipimidate in
Penetration
depth in gelatin (mm)
Penel ra lit) n
0.90 .28
90 85
45
2.00 2.52
60
2.85
67 56 48
120
25
ISO
3.05 3.08
21
7.5 8(1 8.5 9.0
180
3.18
is
9.5
Fixation
lime (mini 0 IS 30
~.
rate (tm miii
00 mM Tris into 10% gelatin gel after 2 h )
Penetration depth in gela tin (mm)
Butter pH
Penetration
rate (gm rnin~)
k
II
0.12
14
0.16 0.20
1.33 .71)
2.13 2.63
8 22
3.05
25
Table 2. Penetration of dimethyladipirnidate in 100 mM Trio pH 9.5 into 10% gelatin gel after 2 Ii Penetration depth in gelatin (mm I
Penetration rate (tm nun ‘(
0.32 2.00
3 17
IS 20
2.72 3.05 2.88
23 25 24
25
2.43
20
Dirncthyladipnntdate concentration )nig. nil)
5 tO
Coefficient of ditlusihility of lixatise )k d il in mm. I in mm I 0.03 0.18 0.25
0.28 ((.26 (1.22
0.24
0.28
Penetration of Adipimidate into Gelatin Gels Table 4. Penetration of 15 mg/mI dimethyladipimidate in Tris pH 9.5 into 10% gelatin gel after 2 h
Buffer
Penetration depth in
Penetration
molarity (mM)
gelatin (mm)
rate (pm min~)
50 100 200 250
2.40 3.05 3.00 2.86
20.0 25.4 25.0 23.8
k 0.22
0.28 0.27
0.26
greater if the molarity is less than 100 mM. If the buffer system is varied, the penetration rate shows also a variation. This variation in the fixative penetration is independent of the buffer penetration (Table 5). With all buffers tested, buffer penetration was more rapid than DMA penetration. DISCUSSION Our results indicate that maximum penetration of DMA into gelatin gels is obtained with fixative concentration 15 mg/ml and high pH (9.5). The same concentration and pH are suggested by other workers (Tzaphlidou, 1987) for optimal cross-linking of collagen by this reagent. However, despite the necessity of high pH to maximize penetration and cross-linking, it cannot be assumed that all biological materials will tolerate such alkaline conditions. Although collagen appeared to be unaffected by the high pH, monkey kidney CVI and COSI cells appeared to suffer major structural changes (Matthopoulos and Tzaphlidou, 1987). It is worth noting that the best light microscopic appearance of these cells after fixation with DMA was achieved with a concentration of DMA of 15 mg/mI. Table 5. Penetration rates of various buffers and of dimethyladipimidate (15 mg/mI) in the same buffers (100 mM, pH 8.0) into 10% gelatin gel after 2 h
Buffer types Tris Tris PBS Tris Earle’s BSS
Penetration rate of buffer (tim mm - t)
Penetration rate of DMA (pm mm - i)
21 28
14 12
24
11
139
The results also show that buffer penetrates faster than dimethyladipimidate. This is in agreement with the work of Millonig (1962) and Trump and Ericsson (1965) on 0s04,who found that the rate of buffer penetration is greater than that of the fixative. However, Coetzee and van der Merwe (1985), using glutaraldehyde as fixative, pointed out that glutaraldehyde penetration is 2—3 times more rapid than buffer penetration. REFERENCES Anderson, W. M. and Fisher, R. R., 1981. The subunit structure ofbovine heart mitochondrial transhydrogenase. Biochim. hioph.v.s. Acta, 635: 194—199. Coetzee, J. and van der Merwe, C. F., 1985. Penetration rate of glutaraldehyde in various buffers into plant tissue and gelatin gels. J. Microsc., 137: 129—136. Davis, P. and Tabor, B. E., 1963. Kinetic study of the crosslinking of gelatin by formaldehyde and glyoxal. J. Pol~m.Sc)., Al: 799--815. Hassell, J. and Hand, A. R., 1974. Tissue fixation with diimidoesters as an alternative to aldehydes. I. Comparison of cross-linking and ultrastructure obtained with dimethylsuberimidate and glutaraldehyde. J. Histochem. Cytochem., 22: 223—239. Henriques, F. and Park, R. B., 1978. Polypeptide crosslinking in chloroplast membranes. Archs Biochem. Biophys., 189: 44—50. Ji, T. H., 1974. Cross-linking of glycolipids in erythrocyte ghost membrane. J. hiol. Chem., 249: 7841—7847. Matthopoulos, D. P. and Tzaphlidou, M., 1987. Tissue culture with of structurefixation obtained withdiimidoesters—I. diimidoesters andComparison formaldehyde. Micron pnicrosc A eta, 18: 273—279. Matthopoulos, D. P. and Tzaphlidou, M., 1988. Tissue culture fixation with diimidoesters—Il. The development of the vimentin type filament network of monkey kidney CVI cells. 19:. 13—17. offixatives. ii Medawar, P. Micron B., 1941.m,cro.sc. The rateActa, ofpenetration R. ,nicrosc. Soc., 61: 41r57. Millonig. G.. 1962. Further observations on a phosphate buffer for osmium solutions in fixation. Fifth mt. Congr. Electron Microsc., 2: 8. R., Mentzer, W. and Lubin, B., Pennathur-Das, R., Heath, 1984. Mechanism of inhibition of sickling by dimethyl adipimidate. Effects of intertetramer cross-linking. Biochim. hiophys. Acta, 791: 259—264. Trump, B. and Ericsson, J. L. E., 1965. The effect of fixative solution on the ultrastructure of cells and tissues. A comparative analysis with particular attention to the proximal convoluted tubule ofthe rat kidney. Lab. Invest.. 14: 1245-1323. Tzaphlidou, M.. 1987. Collagen cross-linking produced by dimethyladipimidate. Its comparison with dimethylsuberimidate. Micron microsc. Acta, 18: 55—58. Tzaphlidou, M. and Chapman, J. A., 1984. A study of positive staining for electron microscopy using collagen as a model system—Ill. The effect of suberimidate fixation. Micron microsc. A eta, 15: 69—76.
0739—6260.88 83.00+1)0(1 Pergamon Press plc
PENETRATION RATE OF DIMETHYLADIPIMIDATE. USE OF GELATIN GELS AS A MODEL SYSTEM MARGARET TZAPHLIDOU*t and DEMETRIOS
P.
MATTHOPOULOSt
Laboratories of *Medical Physics and tGeneral Biology, Medical School, University of loannina, P.O. Box 1186, loannina 45110, Greece (Received 11 March 1988)
Abstract—The rate of dimethyladipimidate penetration Into gelatin gels is influenced primarily by the fixative concentration, pH, temperature and type of buffer. Buffer penetration into the same gels is more rapid than dimethyladipimidate penetration. Index key words: Penetration, dimethyladipimidate, gelatin gels.
INTRODUCTION The bifunctional cross-linking reagent (fixative) dimethyladipimidate (DMA) belongs to the class of diimidoesters and it has been employed to cross-link a variety of proteins (Anderson and Fisher, 1981; Pennathur-Das et al., 1984) and cellular membranes (Ji, 1974; Henriques and Park, 1978). It has also been used in electron and light microscopy to study rat liver (Hassell and Hand, 1974), collagen (Tzaphlidou, 1987) and monkey kidney CV! and COS1 cells (Matthopoulos and Tzaphlidou, 1987, 1988). Even though diimidoesters and particularly dimethyladipimidate have been applied to various biological materials, their penetration rate has not yet been studied. The penetration rate of a fixative is of paramount importance as the depth of penetration is the same as the depth of fixation. Fixative penetration can be calculated by the expression d=k\/t (Medawar, 1941), where d=depth of fixative penetration in mm, = time in mm and k = constant (coefficient of diffusibility of the fixative). The primary purpose underlying the work carried out in the present investigation has been to account for the factors determining the penetration rate of dimethyladipimidate into gelatin gels.
MATERIALS AND METHODS Gelatin solutions were used by Davis and Tabor (1963) and Coetzee and van der Merwe (1985) as a model system to study the penetration rate and the cross-linking effect of aldehydes on a protein. This system allows accurately defined material to be used for analysis and was therefore employed here. The dimethyladipimidate dihydrochloride (DMA) was from Sigma Chemical Co. Fixative solutions were prepared immediately prior to use as described previously (Tzaphlidou, 1987; Tzaphlidou and Chapman, 1984). The buffer types used were: (i) Tris, (ii) Tris Dulbecco’s phosphate buffer saline (PBS) and (iii) Tris Earle’s basal salt solution (BSS). The penetration of DMA and buffer into gelatin was estimated as described by Coetzee and van der Merwe (1985) with minor modifications. Glass tubes (8 mm i.d., 30mm long) were closed at one end with Paraflim and filled with a 10% bacteriological grade aqueous gelatin solution. After cooling, the Parafilm was removed and the tubes were stood in 5 ml fixative solutions in 25 ml glass beakers. After the required fixation time, the cross-linked gelatin was removed by immersing the tubes in hot water at 60°Cfor 2 mm. During this time, the uncrosslinked gelatin is melted and can be poured out. Rinsing of the tubes with water at the same
~ Author to whom correspondence should be sent. 137
I 38
Mirgarcl l 7aphlidou and Demetrto~P. MatthopouIo~
temperature removes the last traces of uncrosslinked gelatin while the cross-linked gelatin forms an insoluble plug in the end of the tube. The length of this plug was measured to the nearest 0.1 mm. The buffer penetration into gelatin was monitored by the change of the indicator color added to the gel. For this purpose, a few drops of a 0.5% aqueous solution of phenol red were added to the gelatin solution before filling the tubes. All values given are the mean of at least four separate repetitions. RESULTS Dimethyladipimidate penetrates into gelatin gel at a rate of 90 ~tm min ‘for the first 10 mm and then drops off (Table I). The penetration rate of DMA into the gel is 21 ~tm min~ for fixation time 2~h which is the time chosen for tissue cross-linking (Tzaphlidou, 1987). The nature of the fixed material plays a role in penetration. The penetration by this diimidoester into 10% gelatin gel that contains O.5% bovine serum albumin is 150 ~immm ‘ in -
.
.
.
.
Table I. Penetration of I ~ mg, nil dimethyladipimidate in 100 mM Tris pH 9.5 into 0% gelatin gel
10 mm. declining to 18 ~tm min in 2 h. When the gelatin is dissolved in Dulbecco’s phosphate buffer saline (pH 7.4) instead of water, the rate of DMA penetration into the gel is 110 ~tm min ‘if thedepthofpenetrationafter 10 ministakeninto consideration. If the penetration rate is calculated for 2 h after contact of the gel with the fixative, the penetration rate is approximately IS j.tm mm In both cases, the values for rates after 2 h are comparable to that obtained for aqueous solution of gelatin (Table I). If, however, the rates after tO mm are taken into consideration, they are high compared with that of Table I. The rate of penetration, and therefore k, depends also on the concentration of DMA (Table 2). An increased concentration of DMA from I mg/mI to 15 mg/mI enhances its rate of penetration while concentrations greater than 15 mg/mI result in a decrease in penetration rate. The value of k is markedly influenced by the pH of the fixative (Table 3). DMA penetrates fasteratroomtemperaturethaninthecold.After 2 h fixation at room temperature, the value of k obtained is 0.28 while at 7C it is 0.24. Penetration is only slightly affected by the molarity of the buffer (Table 4). The effect is -
Table 3. Penetration of IS mg, ml dimethyladipimidate in
Penetration
depth in gelatin (mm)
Penel ra lit) n
0.90 .28
90 85
45
2.00 2.52
60
2.85
67 56 48
120
25
ISO
3.05 3.08
21
7.5 8(1 8.5 9.0
180
3.18
is
9.5
Fixation
lime (mini 0 IS 30
~.
rate (tm miii
00 mM Tris into 10% gelatin gel after 2 h )
Penetration depth in gela tin (mm)
Butter pH
Penetration
rate (gm rnin~)
k
II
0.12
14
0.16 0.20
1.33 .71)
2.13 2.63
8 22
3.05
25
Table 2. Penetration of dimethyladipirnidate in 100 mM Trio pH 9.5 into 10% gelatin gel after 2 Ii Penetration depth in gelatin (mm I
Penetration rate (tm nun ‘(
0.32 2.00
3 17
IS 20
2.72 3.05 2.88
23 25 24
25
2.43
20
Dirncthyladipnntdate concentration )nig. nil)
5 tO
Coefficient of ditlusihility of lixatise )k d il in mm. I in mm I 0.03 0.18 0.25
0.28 ((.26 (1.22
0.24
0.28
Penetration of Adipimidate into Gelatin Gels Table 4. Penetration of 15 mg/mI dimethyladipimidate in Tris pH 9.5 into 10% gelatin gel after 2 h
Buffer
Penetration depth in
Penetration
molarity (mM)
gelatin (mm)
rate (pm min~)
50 100 200 250
2.40 3.05 3.00 2.86
20.0 25.4 25.0 23.8
k 0.22
0.28 0.27
0.26
greater if the molarity is less than 100 mM. If the buffer system is varied, the penetration rate shows also a variation. This variation in the fixative penetration is independent of the buffer penetration (Table 5). With all buffers tested, buffer penetration was more rapid than DMA penetration. DISCUSSION Our results indicate that maximum penetration of DMA into gelatin gels is obtained with fixative concentration 15 mg/ml and high pH (9.5). The same concentration and pH are suggested by other workers (Tzaphlidou, 1987) for optimal cross-linking of collagen by this reagent. However, despite the necessity of high pH to maximize penetration and cross-linking, it cannot be assumed that all biological materials will tolerate such alkaline conditions. Although collagen appeared to be unaffected by the high pH, monkey kidney CVI and COSI cells appeared to suffer major structural changes (Matthopoulos and Tzaphlidou, 1987). It is worth noting that the best light microscopic appearance of these cells after fixation with DMA was achieved with a concentration of DMA of 15 mg/mI. Table 5. Penetration rates of various buffers and of dimethyladipimidate (15 mg/mI) in the same buffers (100 mM, pH 8.0) into 10% gelatin gel after 2 h
Buffer types Tris Tris PBS Tris Earle’s BSS
Penetration rate of buffer (tim mm - t)
Penetration rate of DMA (pm mm - i)
21 28
14 12
24
11
139
The results also show that buffer penetrates faster than dimethyladipimidate. This is in agreement with the work of Millonig (1962) and Trump and Ericsson (1965) on 0s04,who found that the rate of buffer penetration is greater than that of the fixative. However, Coetzee and van der Merwe (1985), using glutaraldehyde as fixative, pointed out that glutaraldehyde penetration is 2—3 times more rapid than buffer penetration. REFERENCES Anderson, W. M. and Fisher, R. R., 1981. The subunit structure ofbovine heart mitochondrial transhydrogenase. Biochim. hioph.v.s. Acta, 635: 194—199. Coetzee, J. and van der Merwe, C. F., 1985. Penetration rate of glutaraldehyde in various buffers into plant tissue and gelatin gels. J. Microsc., 137: 129—136. Davis, P. and Tabor, B. E., 1963. Kinetic study of the crosslinking of gelatin by formaldehyde and glyoxal. J. Pol~m.Sc)., Al: 799--815. Hassell, J. and Hand, A. R., 1974. Tissue fixation with diimidoesters as an alternative to aldehydes. I. Comparison of cross-linking and ultrastructure obtained with dimethylsuberimidate and glutaraldehyde. J. Histochem. Cytochem., 22: 223—239. Henriques, F. and Park, R. B., 1978. Polypeptide crosslinking in chloroplast membranes. Archs Biochem. Biophys., 189: 44—50. Ji, T. H., 1974. Cross-linking of glycolipids in erythrocyte ghost membrane. J. hiol. Chem., 249: 7841—7847. Matthopoulos, D. P. and Tzaphlidou, M., 1987. Tissue culture with of structurefixation obtained withdiimidoesters—I. diimidoesters andComparison formaldehyde. Micron pnicrosc A eta, 18: 273—279. Matthopoulos, D. P. and Tzaphlidou, M., 1988. Tissue culture fixation with diimidoesters—Il. The development of the vimentin type filament network of monkey kidney CVI cells. 19:. 13—17. offixatives. ii Medawar, P. Micron B., 1941.m,cro.sc. The rateActa, ofpenetration R. ,nicrosc. Soc., 61: 41r57. Millonig. G.. 1962. Further observations on a phosphate buffer for osmium solutions in fixation. Fifth mt. Congr. Electron Microsc., 2: 8. R., Mentzer, W. and Lubin, B., Pennathur-Das, R., Heath, 1984. Mechanism of inhibition of sickling by dimethyl adipimidate. Effects of intertetramer cross-linking. Biochim. hiophys. Acta, 791: 259—264. Trump, B. and Ericsson, J. L. E., 1965. The effect of fixative solution on the ultrastructure of cells and tissues. A comparative analysis with particular attention to the proximal convoluted tubule ofthe rat kidney. Lab. Invest.. 14: 1245-1323. Tzaphlidou, M.. 1987. Collagen cross-linking produced by dimethyladipimidate. Its comparison with dimethylsuberimidate. Micron microsc. Acta, 18: 55—58. Tzaphlidou, M. and Chapman, J. A., 1984. A study of positive staining for electron microscopy using collagen as a model system—Ill. The effect of suberimidate fixation. Micron microsc. A eta, 15: 69—76.