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Tryptic cleavage of IgD at elevated temperature and isolation of a Fc-like fragment in high yield
~nmunoehemistry, 1975, Vol, 12, pp. 685 689. Pergamon Press.
Printed in Great Britain
TRYPTIC CLEAVAGE OF IgD AT ELEVATED TEMPERATURE A N D ISOLATION OF A Fc-LIKE FRAGMENT IN HIGH YIELD* M I C H A E L W O L C O T T t , D O U G L A S G. F R E E M A N , R A L P H E. S C H R O H E N L O H E R , W I L L I A M H A M M A C K and J. C L A U D E B E N N E T T Departments of Microbiology and Medicine University of Alabama in Birmingham University Station, Birmingham, Alabama 35294, U.S.A. and Veterans Administration Hospital, Birmingham, Alabama 35233, U.S.A. (First received 11 January 1974; in revised form 12 November 1974)
Abstract--Digestion of IgD with trypsin at 56°C for 4 min has been shown to be an effective method for the production of homogeneous Fc-like component in high yield. The first two residues of this fragment were found to be Thr-Ala. Because of rapid and continuous degradation this method does not allow production of Fab-like components in significant quantities.
INTRODUCTION
MATERIALS AND METHODS
~uccessful analysis of the primary structure of any :omplex protein depends upon the specific and repro~ucible cleavage of such molecules into defined sub:omponents. This has been particularly useful in the ~tudy of immunoglobulin structure, particularly in the tbility of various enzymes to produce Fab-like and Fc-like fragments. Most immunoglobulins, regardless )f their class, have in fact been found to be susceptible :o digestion with either papain, trypsin, or pepsin to live such defined and reproducible fragments (Porter, 1959; Miller and Metzger, 1966; Mihaesco and Selig~ann, 1968; Chen et al., 1969). This paper reports a method of enzymatic cleavage )f the IgD molecule such that a Fc-like fragment suittble ~for sequence analysis could be isolated. It would ~e desirable for such a fragment to be produced with t high degree of homogeneity and to contain only t single N-terminal residue. Proteolytic cleavage pos;essing this degree of specificity would be found most ikely with trypsin. However, limited digestion with :rypsin is somewhat difficult to achieve and therefore m approach was taken similar to that reported by Plaut and Tomasi (1970) in the study of IgM. In this nethod digestion was carried out at high tempera:ures, that is above 50 degrees, in order to achieve fignificant yields of fragments with good reproducibilty. The kinetics of digestion of IgD by trypsin at 56°C md isolation of a Fc-like fragment are given in this ~aper.
Materials Immunoglobulin D~. was isolated from the serum of a patient (HEN) with multiple myeloma by a method similar to that described by Spiegelberg et al. (1970). In brief, the serum was dialyzed in the cold against buffer, 0.015 M Tris, pH 8. The dialyzed serum was then applied to a column of DEAE-Sephadex A-50 (Pharmacia, Piscataway, N.J.) equilibrated with the same buffer. The column was eluted with this same buffer until elution of u.v. absorbing material was complete. At this point a linear salt gradient was started. The gradient was: start buffer, 0.015 M Tris, pH 8-0; limit buffer, 0.015M Tris, 0.75M NaC1, pH 8.0. The fractions containing IgD were located by analysis of every odd fraction by Oucherlony method of double diffusion in agar using commercially available (Hyland Labs, Costa Mesa, CA. or Meloy Labs, SpFingfield, VA.) antiIgD sera produced in goats. Additional purification was carried out on Sephadex G-200 in 0.01M Tris, 0.14M NaCI, pH 7.4. All buffers in these procedures contained 10 -3 M 6-aminocaproic acid to reduce endogenous proteolytic cleavage of IgD. Trypsin (TPCK) and soy bean trypsin inhibitor were obtained from Worthington Biochemical Company, Freehold, New Jersey.
* This investigation was supported by United States ?ublic Health Service Research Grant, ROICAI6322. t To whom reprint requests should be sent.
Enzymatic dioestion The IgD sample and the trypsin were prepared separately in pH 8.0 buffer 0.005 M CaClz) and equilibrated in a water bath at 56° prior to mixing. The final concentration of the reactants (weight/volume) was 2 per cent IgD and 0.08 per cent trypsin. Samples were removed at various time intervals and placed in tubes at 0 ° containing soy bean trypsin inhibitor. In each case there was a 1.5 fold excess of trypsin inhibitor (wt/wt) relative to the amount of trypsin present.
Analytical methods SDS polyacrylamide disc electrophoresis was carried out according to the method of Maizel (1966) in 0.1 M phos685
686
M. WOLCOTT et al.
phate buffer pH 7.2 with 0.1% sodium lauryl sulfate and 1 M urea. Immunoelectrophoresis ffas carried out by the method of Seheidegger (1955) in 0.2 per cent agarose and veronal buffer (ionic strength 0"05, pH 8.2).
The dansyl amino acids librated were identified by chro matography on polyamide thin layer plates (Cheng Chil Trading Co., Ltd., Taiwan; sold by Gallard-Schlesinger New York) as described by Woods and Wang (1967).
Ultracentrifuoation
RESULTS
Sedimentation velocity measurements were made on samples of the digestion mixture, taken at various times and isolated Fc fragment, using a Beckman Model E analytical ultracentrifuge equipped with schliern optics. The sedimentation rate was determined for the Fc fragment according to the method of Sehachman (1959). Molecular weight of the Fc fragment was determined in the ultracentrifuge by sedimentation equilibrium using absorption optics and a photoelectric scanner (Lamers et al., 1963). The Fc fragment was dissolved in saline at protein concentrations of 0'23 and 0-43 mg/ml. Analyses were performed at 9945 rev/min using a sample column height of 3mm. The light source monochromator was set at 280 nm and the temperature was regulated at 20°C. Equilibrium was judged to be established when there was no change in the slope of the logarithm of the absorbance (A) plotted against the square of the distance from the center of rotation (r) over a minimum of 3 hr. In calculating the molecular weight, the partial specific volume was assumed to be 0.73 ml/g (Sehachman, 1959).
Experiments to determine the optimal time foJ digestion of IgD by trypsin at 56°C were undertaker first. The most favorable time was considered to b~ that point at which the maximum a m o u n t of IgE had been degraded but with a minimal amount o: secondary fragmentation. The kinetics of digestior were followed by examining samples taken from the reaction mixture at 2 min intervals up to 20 min witl: additional samples at 30 and 60 rain. Each of thes~ was analyzed by immunoelectrophoresis and polyacrylamide disc gel electrophoresis. Figure 1 shows the electrophoretic patterns of samples taken at 0, 2, 4 and 10min. It should be noted from SDS gels thai exposure of IgD to trypsin for 2 min results in the total cleavage of the molecule as evidenced by the disappearance of the band corresponding to the starting material. At the same time faster migrating components appear. Longer periods of digestion result in additional fragmentation of the molecule as illustrated by the appearance of lower bands on gels for material taken at 4 min and later.
N-terminal amino acid analysis
Analysis of the N-terminal amino acids was done by the dansyl-Edman procedure described by Grey (1967).
Anode
Cathode Anode
Cathode o
0
b
o
2
b
a
4
b
a=Anti
sera
to human
~ chain
b--Anti
sera
to human
X chain
a
I0
b
Fig. l. Top: SDS polyacrylamide gel electrophoresis of samples taken at the indicated times from a 56°C tryptic digestion of IgD. Conditions for digestion are given in Materials and Methods. Bottom: Immunoelectrophoretic patterns of the same samples.
Trypt~Cleavage of lgD
Time
687
of
hydrolysis
E
I0 rain
4 rain
2 min
Omin
Sedimentation
--~
Fig. 2. Ultraeentrifugal analysis of samples taken at the indicated times from the 56°C tryptic digestion of IgD. The samples were diluted to a concentration equivalent to 5 nag IgD/ml. The photographs were taken after 64rain at 56,100 rev/min at a phase plate angle of 60°. Sedimentation proceeds from left to right. Immunoelectrophoresis samples of the digestion nixture were examined by analyses for both delta and ambda chains. Digestion of IgD for 2 min and longer esults in the formation of a component, which conains only t5 chain determinants and has rapid cathode migration. Although the IgD has been completely Legraded after 2 min, there is a component which aaintains both heavy and light chain determinants. ?his double reacting component, therefore, must be • fragment smaller than the starting parent molecule. ~bsence of any intact IgD in the digested samples confirmed by the ultracentrifugal sedimentation .nalysis (Fig. 2). Digestion for 2 min resulted in the leavage of the IgD molecule to fragments of nearly qual size. Longer periods of digestion produce dditional fragments of smaller size as evidenced ~y the increased amounts of slower sedimenting aaterial (see Fig. 2). This corresponds to the more omplicated disc gel patterns for the longer periods pf digestion. Even though disappearance of starting material was ~oted at 2 min of digestion, a more stable pattern ,f four major components ertierged at 4 min. In order
to achieve better reproducibility and to capitalize upon the more stable pattern of fragmentation this latter time was taken as the point for further analysis of the major resulting components. Two hundred milligrams of IgD were digested with trypsin for 4 min at 56°C. After cessation of the reaction with soy bean trypsin inhibitor the digestion mixture was fractionated on DEAE Sephadex A-50 (Fig. 3). Peak I gave multiple bands on disc electrophoresis, eluted from Sephadex G-200 indicating a molecular weight of approximately 48,000 and contained only lambda chain determinants. Although the products were not homogeneous, the size and immunochemical characteristics are suggestive of a Fab-like fragment. Peak II did not react with either anti-2 nor anti-6 chain antisera. This material on further purification from G-100 Sephadex had an elution position indicating a molecular weight of approximately 18,000. Peak III contained determinants of both the 2 and 6 chains. Due to small amounts of this fraction further attempts of purification were unsuccessful. However, the apparent molecular weight on SDS gels was found to be high, that is, greater than 100,000.
688
M. W O L C O T T et al.
r~r
im ~
I 1I
1.2 ~ I'1
D
1.0
m
0"9-0'8--
0
0'8
/
0 -7
0.7
0 .6
0-6
o "5
0.5
e
o
0"4
e,
0
0.3
o
0
0.2
"~
0
0.1
~
.4
0
20
40
60
Tube
80
I00
120
140
160
~_
180
number
Fig. 3. Fractionation of 4 min 56°C tryptic digestion of IgD on a DEAE-Sephadex A-50 anion exchange column (2.5 x 45 cm). After fifty 5 ml fractions had been eluted with the starting buffer (0"05 M Tris, pH 8 ) a linear salt gradient was begun. The gradient was: start buffer~0.05 M Tris, pH 8; limit buffer-0"05 M Tris, 0.7 M NaC1, pH 8"0.
Peak IV was examined by the Oucherlony technique of double diffusion in agar. Plates were developed with anti-2 chain and anti-6 chain antisera. Only the anti-6 chain sera reacted with this material. Peak IV was run on immunoelectrophoresis and again
16 m i n
reacted only with anti-6 chain sera. This material was shown to be identical with the cathodic migrating material seen in Fig. 1. Analysis by disc gel electrophoresis (insert in Fig, 3) and analytical ultracentrifugation (Fig. 4) indicated
64min Time
of
1 0 4 min
sedimentotion
Fig. 4. Sedimentation velocity experiment in saline of isolated Fc fragment, 5.7 mg/ml. The photographs were taken at the indicated times after reaching a speed of 56,100rev/min at a phase plate angle of 60 ° and temperature of 20°C. Sedimentation proceeds from left to right.
Tryptic Cleavage of IgD mt this fraction was homogeneous. The sedilentation c o n s t a n t (S20,w) was found to be 3"5 sec. 'he molecular weight of the fragment was determined y sedimentation equilibrium. The calculated values 'ere 64,000 and 64,100 for the 0-23 and 0"43 mg/ml tmples respectively. Both samples gave linear plots f log A vs r 2. The latter observation provided further vidence that this fraction was homogeneous. These ata suggest that this material is a Fc-like component f IgD. Calculations on a weight basis indicate a yield f 80-85 per cent. N-terminal amino acid analysis was carried out on ae isolated Fc fragment by the dansyl-Edman proedure. The Fc was found to contain a single N-terainal residue which was threonine. A second cycle evealed alanine at the second position. DISCUSSION Digestion of immunoglobulin D with trypsin at 6°C for 4 min results in the production of a homoeneous Fc-like component which can be isolated in igh yield. The identification of this component as eing Fc-like was established by the facts that it contins only delta chain determinants and has a molecutr weight of approximately 64,000. Its homogeneity 1as established by analysis in polyacrylamide disc gel lectrophoresis, immunoelectrophoresis, ultracentrifual sedimentation and N-terminal group analysis. It tould appear that this material is ideal for further ,rimary amino acid structural studies.
689
By contrast the Fab-like component of IgD has not been isolated in homogeneous form. In fact it appears to be susceptible to continual digestion under the condition of these experiments. Chromatographic analysis of samples digested for other times indicated that peaks, I, II and III (Fig. 3) undergo interchanges relative to the amounts present. Such an observation is compatible with instability of the Fab-like components and their continual fragmentation. Peak IV, i.e. the Fc-like fragment, tends to be relatively stable. REFERENCES
Cben J. P., Reichlin M. and Tomasi T. B., Jr. (1967) Biochemistry 8, 2246. Grey W. R. (1967) Methods in Enzymology 11, 139. Lamers K., Putney F., Steinberg I. Z. and Schachman H. K. (1963) Arch. Biochem. Biophys. 103, 379. Maizel J. V., Jr. (1966) Science 151, 988. Mihaesco C. and Seligmann M. (1967) J. exp Med. 127, 431. Miller F. and Metzger H. (1966) J. biol. Chem. 241, 1732. Plaut A. G. and Tomasi T. B., Jr. (1970) Proc. natn Acad. Sci. 65, 318. Porter R. R. (1959) Biochem. J. 73, 119. Schachman H. K. (1959) Ultracentrifuoation in Biochemistry. Academic Press, New York, N.Y. Scheidegger J. J. (1955) Int. Archs Allergy appl. lmmun. 7, 103. Spiegelberg H. L., Prahl J. W. and Greg H. M. (1970) Biochemistry 9, 2115. Woods K. R. and Wang K. (1967) Biochem. biophys. Acta, 133, 369.
Printed in Great Britain
TRYPTIC CLEAVAGE OF IgD AT ELEVATED TEMPERATURE A N D ISOLATION OF A Fc-LIKE FRAGMENT IN HIGH YIELD* M I C H A E L W O L C O T T t , D O U G L A S G. F R E E M A N , R A L P H E. S C H R O H E N L O H E R , W I L L I A M H A M M A C K and J. C L A U D E B E N N E T T Departments of Microbiology and Medicine University of Alabama in Birmingham University Station, Birmingham, Alabama 35294, U.S.A. and Veterans Administration Hospital, Birmingham, Alabama 35233, U.S.A. (First received 11 January 1974; in revised form 12 November 1974)
Abstract--Digestion of IgD with trypsin at 56°C for 4 min has been shown to be an effective method for the production of homogeneous Fc-like component in high yield. The first two residues of this fragment were found to be Thr-Ala. Because of rapid and continuous degradation this method does not allow production of Fab-like components in significant quantities.
INTRODUCTION
MATERIALS AND METHODS
~uccessful analysis of the primary structure of any :omplex protein depends upon the specific and repro~ucible cleavage of such molecules into defined sub:omponents. This has been particularly useful in the ~tudy of immunoglobulin structure, particularly in the tbility of various enzymes to produce Fab-like and Fc-like fragments. Most immunoglobulins, regardless )f their class, have in fact been found to be susceptible :o digestion with either papain, trypsin, or pepsin to live such defined and reproducible fragments (Porter, 1959; Miller and Metzger, 1966; Mihaesco and Selig~ann, 1968; Chen et al., 1969). This paper reports a method of enzymatic cleavage )f the IgD molecule such that a Fc-like fragment suittble ~for sequence analysis could be isolated. It would ~e desirable for such a fragment to be produced with t high degree of homogeneity and to contain only t single N-terminal residue. Proteolytic cleavage pos;essing this degree of specificity would be found most ikely with trypsin. However, limited digestion with :rypsin is somewhat difficult to achieve and therefore m approach was taken similar to that reported by Plaut and Tomasi (1970) in the study of IgM. In this nethod digestion was carried out at high tempera:ures, that is above 50 degrees, in order to achieve fignificant yields of fragments with good reproducibilty. The kinetics of digestion of IgD by trypsin at 56°C md isolation of a Fc-like fragment are given in this ~aper.
Materials Immunoglobulin D~. was isolated from the serum of a patient (HEN) with multiple myeloma by a method similar to that described by Spiegelberg et al. (1970). In brief, the serum was dialyzed in the cold against buffer, 0.015 M Tris, pH 8. The dialyzed serum was then applied to a column of DEAE-Sephadex A-50 (Pharmacia, Piscataway, N.J.) equilibrated with the same buffer. The column was eluted with this same buffer until elution of u.v. absorbing material was complete. At this point a linear salt gradient was started. The gradient was: start buffer, 0.015 M Tris, pH 8-0; limit buffer, 0.015M Tris, 0.75M NaC1, pH 8.0. The fractions containing IgD were located by analysis of every odd fraction by Oucherlony method of double diffusion in agar using commercially available (Hyland Labs, Costa Mesa, CA. or Meloy Labs, SpFingfield, VA.) antiIgD sera produced in goats. Additional purification was carried out on Sephadex G-200 in 0.01M Tris, 0.14M NaCI, pH 7.4. All buffers in these procedures contained 10 -3 M 6-aminocaproic acid to reduce endogenous proteolytic cleavage of IgD. Trypsin (TPCK) and soy bean trypsin inhibitor were obtained from Worthington Biochemical Company, Freehold, New Jersey.
* This investigation was supported by United States ?ublic Health Service Research Grant, ROICAI6322. t To whom reprint requests should be sent.
Enzymatic dioestion The IgD sample and the trypsin were prepared separately in pH 8.0 buffer 0.005 M CaClz) and equilibrated in a water bath at 56° prior to mixing. The final concentration of the reactants (weight/volume) was 2 per cent IgD and 0.08 per cent trypsin. Samples were removed at various time intervals and placed in tubes at 0 ° containing soy bean trypsin inhibitor. In each case there was a 1.5 fold excess of trypsin inhibitor (wt/wt) relative to the amount of trypsin present.
Analytical methods SDS polyacrylamide disc electrophoresis was carried out according to the method of Maizel (1966) in 0.1 M phos685
686
M. WOLCOTT et al.
phate buffer pH 7.2 with 0.1% sodium lauryl sulfate and 1 M urea. Immunoelectrophoresis ffas carried out by the method of Seheidegger (1955) in 0.2 per cent agarose and veronal buffer (ionic strength 0"05, pH 8.2).
The dansyl amino acids librated were identified by chro matography on polyamide thin layer plates (Cheng Chil Trading Co., Ltd., Taiwan; sold by Gallard-Schlesinger New York) as described by Woods and Wang (1967).
Ultracentrifuoation
RESULTS
Sedimentation velocity measurements were made on samples of the digestion mixture, taken at various times and isolated Fc fragment, using a Beckman Model E analytical ultracentrifuge equipped with schliern optics. The sedimentation rate was determined for the Fc fragment according to the method of Sehachman (1959). Molecular weight of the Fc fragment was determined in the ultracentrifuge by sedimentation equilibrium using absorption optics and a photoelectric scanner (Lamers et al., 1963). The Fc fragment was dissolved in saline at protein concentrations of 0'23 and 0-43 mg/ml. Analyses were performed at 9945 rev/min using a sample column height of 3mm. The light source monochromator was set at 280 nm and the temperature was regulated at 20°C. Equilibrium was judged to be established when there was no change in the slope of the logarithm of the absorbance (A) plotted against the square of the distance from the center of rotation (r) over a minimum of 3 hr. In calculating the molecular weight, the partial specific volume was assumed to be 0.73 ml/g (Sehachman, 1959).
Experiments to determine the optimal time foJ digestion of IgD by trypsin at 56°C were undertaker first. The most favorable time was considered to b~ that point at which the maximum a m o u n t of IgE had been degraded but with a minimal amount o: secondary fragmentation. The kinetics of digestior were followed by examining samples taken from the reaction mixture at 2 min intervals up to 20 min witl: additional samples at 30 and 60 rain. Each of thes~ was analyzed by immunoelectrophoresis and polyacrylamide disc gel electrophoresis. Figure 1 shows the electrophoretic patterns of samples taken at 0, 2, 4 and 10min. It should be noted from SDS gels thai exposure of IgD to trypsin for 2 min results in the total cleavage of the molecule as evidenced by the disappearance of the band corresponding to the starting material. At the same time faster migrating components appear. Longer periods of digestion result in additional fragmentation of the molecule as illustrated by the appearance of lower bands on gels for material taken at 4 min and later.
N-terminal amino acid analysis
Analysis of the N-terminal amino acids was done by the dansyl-Edman procedure described by Grey (1967).
Anode
Cathode Anode
Cathode o
0
b
o
2
b
a
4
b
a=Anti
sera
to human
~ chain
b--Anti
sera
to human
X chain
a
I0
b
Fig. l. Top: SDS polyacrylamide gel electrophoresis of samples taken at the indicated times from a 56°C tryptic digestion of IgD. Conditions for digestion are given in Materials and Methods. Bottom: Immunoelectrophoretic patterns of the same samples.
Trypt~Cleavage of lgD
Time
687
of
hydrolysis
E
I0 rain
4 rain
2 min
Omin
Sedimentation
--~
Fig. 2. Ultraeentrifugal analysis of samples taken at the indicated times from the 56°C tryptic digestion of IgD. The samples were diluted to a concentration equivalent to 5 nag IgD/ml. The photographs were taken after 64rain at 56,100 rev/min at a phase plate angle of 60°. Sedimentation proceeds from left to right. Immunoelectrophoresis samples of the digestion nixture were examined by analyses for both delta and ambda chains. Digestion of IgD for 2 min and longer esults in the formation of a component, which conains only t5 chain determinants and has rapid cathode migration. Although the IgD has been completely Legraded after 2 min, there is a component which aaintains both heavy and light chain determinants. ?his double reacting component, therefore, must be • fragment smaller than the starting parent molecule. ~bsence of any intact IgD in the digested samples confirmed by the ultracentrifugal sedimentation .nalysis (Fig. 2). Digestion for 2 min resulted in the leavage of the IgD molecule to fragments of nearly qual size. Longer periods of digestion produce dditional fragments of smaller size as evidenced ~y the increased amounts of slower sedimenting aaterial (see Fig. 2). This corresponds to the more omplicated disc gel patterns for the longer periods pf digestion. Even though disappearance of starting material was ~oted at 2 min of digestion, a more stable pattern ,f four major components ertierged at 4 min. In order
to achieve better reproducibility and to capitalize upon the more stable pattern of fragmentation this latter time was taken as the point for further analysis of the major resulting components. Two hundred milligrams of IgD were digested with trypsin for 4 min at 56°C. After cessation of the reaction with soy bean trypsin inhibitor the digestion mixture was fractionated on DEAE Sephadex A-50 (Fig. 3). Peak I gave multiple bands on disc electrophoresis, eluted from Sephadex G-200 indicating a molecular weight of approximately 48,000 and contained only lambda chain determinants. Although the products were not homogeneous, the size and immunochemical characteristics are suggestive of a Fab-like fragment. Peak II did not react with either anti-2 nor anti-6 chain antisera. This material on further purification from G-100 Sephadex had an elution position indicating a molecular weight of approximately 18,000. Peak III contained determinants of both the 2 and 6 chains. Due to small amounts of this fraction further attempts of purification were unsuccessful. However, the apparent molecular weight on SDS gels was found to be high, that is, greater than 100,000.
688
M. W O L C O T T et al.
r~r
im ~
I 1I
1.2 ~ I'1
D
1.0
m
0"9-0'8--
0
0'8
/
0 -7
0.7
0 .6
0-6
o "5
0.5
e
o
0"4
e,
0
0.3
o
0
0.2
"~
0
0.1
~
.4
0
20
40
60
Tube
80
I00
120
140
160
~_
180
number
Fig. 3. Fractionation of 4 min 56°C tryptic digestion of IgD on a DEAE-Sephadex A-50 anion exchange column (2.5 x 45 cm). After fifty 5 ml fractions had been eluted with the starting buffer (0"05 M Tris, pH 8 ) a linear salt gradient was begun. The gradient was: start buffer~0.05 M Tris, pH 8; limit buffer-0"05 M Tris, 0.7 M NaC1, pH 8"0.
Peak IV was examined by the Oucherlony technique of double diffusion in agar. Plates were developed with anti-2 chain and anti-6 chain antisera. Only the anti-6 chain sera reacted with this material. Peak IV was run on immunoelectrophoresis and again
16 m i n
reacted only with anti-6 chain sera. This material was shown to be identical with the cathodic migrating material seen in Fig. 1. Analysis by disc gel electrophoresis (insert in Fig, 3) and analytical ultracentrifugation (Fig. 4) indicated
64min Time
of
1 0 4 min
sedimentotion
Fig. 4. Sedimentation velocity experiment in saline of isolated Fc fragment, 5.7 mg/ml. The photographs were taken at the indicated times after reaching a speed of 56,100rev/min at a phase plate angle of 60 ° and temperature of 20°C. Sedimentation proceeds from left to right.
Tryptic Cleavage of IgD mt this fraction was homogeneous. The sedilentation c o n s t a n t (S20,w) was found to be 3"5 sec. 'he molecular weight of the fragment was determined y sedimentation equilibrium. The calculated values 'ere 64,000 and 64,100 for the 0-23 and 0"43 mg/ml tmples respectively. Both samples gave linear plots f log A vs r 2. The latter observation provided further vidence that this fraction was homogeneous. These ata suggest that this material is a Fc-like component f IgD. Calculations on a weight basis indicate a yield f 80-85 per cent. N-terminal amino acid analysis was carried out on ae isolated Fc fragment by the dansyl-Edman proedure. The Fc was found to contain a single N-terainal residue which was threonine. A second cycle evealed alanine at the second position. DISCUSSION Digestion of immunoglobulin D with trypsin at 6°C for 4 min results in the production of a homoeneous Fc-like component which can be isolated in igh yield. The identification of this component as eing Fc-like was established by the facts that it contins only delta chain determinants and has a molecutr weight of approximately 64,000. Its homogeneity 1as established by analysis in polyacrylamide disc gel lectrophoresis, immunoelectrophoresis, ultracentrifual sedimentation and N-terminal group analysis. It tould appear that this material is ideal for further ,rimary amino acid structural studies.
689
By contrast the Fab-like component of IgD has not been isolated in homogeneous form. In fact it appears to be susceptible to continual digestion under the condition of these experiments. Chromatographic analysis of samples digested for other times indicated that peaks, I, II and III (Fig. 3) undergo interchanges relative to the amounts present. Such an observation is compatible with instability of the Fab-like components and their continual fragmentation. Peak IV, i.e. the Fc-like fragment, tends to be relatively stable. REFERENCES
Cben J. P., Reichlin M. and Tomasi T. B., Jr. (1967) Biochemistry 8, 2246. Grey W. R. (1967) Methods in Enzymology 11, 139. Lamers K., Putney F., Steinberg I. Z. and Schachman H. K. (1963) Arch. Biochem. Biophys. 103, 379. Maizel J. V., Jr. (1966) Science 151, 988. Mihaesco C. and Seligmann M. (1967) J. exp Med. 127, 431. Miller F. and Metzger H. (1966) J. biol. Chem. 241, 1732. Plaut A. G. and Tomasi T. B., Jr. (1970) Proc. natn Acad. Sci. 65, 318. Porter R. R. (1959) Biochem. J. 73, 119. Schachman H. K. (1959) Ultracentrifuoation in Biochemistry. Academic Press, New York, N.Y. Scheidegger J. J. (1955) Int. Archs Allergy appl. lmmun. 7, 103. Spiegelberg H. L., Prahl J. W. and Greg H. M. (1970) Biochemistry 9, 2115. Woods K. R. and Wang K. (1967) Biochem. biophys. Acta, 133, 369.