A New Method for Determination of Alpha-1 antitrypsin Phenotypes Using Isoelectric Focusing on Polyacrylamide Gel Slabs

Transcription

1 A New Method for Determination of Alpha-1 antitrypsin Phenotypes Using Isoelectric Focusing on Polyacrylamide Gel Slabs ROBERT C. ALLEN, PH.D., RUSSELL A. HARLEY, RICHARD C. TALAMO, M.D. M.D., Department of Pathology, Medical University of South Carolina, Charleston, South Carolina, and Children's Service, Massachusetts General Hospital, Boston, Massachusetts ABSTRACT Allen, Robert C, Harley, Russell A., and Talamo, Richard C: A new method for determination of alpha-1-antitrypsin phenotypes using isoelectric focusing on polyacrylamide gel slabs. Am. J. Clin. Pathol. 62: , Phenotyping methods for alpha-1-antitrypsin were developed utilizing isoelectric focusing on 1 mm. acrylamide gel slabs. Alpha-1-antitrypsin proteins have isoelectric points in the ph region from 4.2 to Utilizing ampholines with a ph range of or 3-6, the proteinase inhibitor (Pi) phenotypes containing the more common M, A, S, F alleles were found to be readily identifiable and may be studied both qualitatively and quantitatively by this method. Protein distributions of the various Pi allele products as illustrated by densitometric traces show patterns similar to those found in the conventional two-dimensional acid starch gel crossing antigen-antibody electrophoresis. The high resolving ability of isoelectric focusing indicated the presence of M allele-like products in FZ, SS, and ZZ Pi types. The total procedure time was reduced from VA days to 4 hours. The potential resolving power of this method appears sufficient to identify also the untested remaining alleles. (Key words: Antitrypsin phenotyping; Isoelectric focusing.) SINCE Laurell and Eriksson 9 described an phenotypes have been described. 8 While association between emphysema and an extensive research has been carried out inherited plasma protein abnormality, over the last 10 years on the association of alpha-1-antitrypsin deficiency, some 11 these deficiencies and the possible relaalleles and 21 proteinase inhibitor (Pi) tionship of Pi phenotypes to disease, a marked deterrent to this endeavor has Received May 15, 1974; revised June 24, 1974; been the complexity of procedures for accepted For publication June 24, accurate phenotyping. Supported by S.C. Institutional Grant # \ i A401, USDA-ARS Cooperative Research Agreement Presently, the most reliable phenotyp- # , and U.S.P.H.S. Contract i n g method requires a two-step procedure #712217, National Heart and Lung Institute. i- i i i i i of a o d starch Address reprint requests to Dr. Allen, Medical g el electrophoresis in com- University of South Carolina. bination with subsequent crossing 732

2 December 1974 PI PHENOTYPING BY ISOELECTRIC FOCUSING 733 antigen-antibody electrophoresis on agar gel plates. This procedure requires a day and a half to complete and 10-20% of the sample may have to be repeated if the antibody concentration in the second step is improperly chosen. 13 Due to the considerable expertise required for the separation and resultant pattern interpretation, Pi phenotyping is generally restricted to a limited number of laboratories. Thus, a simple rapid method of phenotyping is essential to catalog and elucidate more fully the relationships of the Pi phenotypes to disease or to a predisposition to disease. To date, methods of electrophoresis other than acid starch gel have been reported to be unable to resolve the various Pi allele products. In sieving media such as polyacrylamide gel with discontinuous buffer of alkaline running ph's of 9.0, Maurer and Allen 11 have shown, using two-dimensional technics, that human alpha-1-antitrypsin migrates with the albumin, while Myerowitz and associates 12 have similarly shown that one species of mouse alpha-1-antitrypsin migrates with the albumin. On the other hand, two-dimensional electrophoresis with the first dimension on cellulose acetate and the second by isoelectric focusing on polyacrylamide gel slabs indicates that the alpha-1 globulins may be separated from the albumin. 10 Since the isoelectric point of the alpha-1 globulin region is at a ph of and that of albumin, ph 4.9, 5 a suitable ph gradient should have the capability of separating the alpha-1-antitrypsin Pi phenotypes. We therefore undertook to develop a rapid single-step system for Pi phenotyping utilizing isoelectric focusing on polyacrylamide gel slabs. Materials and Methods Trypsin inhibitory capacity: Serum trypsin inhibitory capacity was determined photometrically by measurement of N-benzoyl-DL-arginine-p-nitroanilide BAPNA hydrolysis after the method of Laurell and Eriksson. 9 Radial immunodiffusion assay: Radial immunodiffusion kits for measurement of serum A-l-AT concentration were obtained from Miles-Pentex Laboratories, Kankakee, Illinois. Quantification was performed against known standards utilizing 5 fil. of serum and measuring the antigen-antibody ring after incubation for 24 hours at room temperature. Acid starch gel electrophoresis: Acid starch gel electrophoresis was employed as described by Fagerhol. 6 Crossing antigen -antibody electrophoresis was performed on antibody containing agarose gels according to the method of Laurell. 10 Isoelectric Focusing Isoelectric focusing was carried out in an LKB Multiphor electrophoresis system modified to take 1-mm.-thick acrylamide gel slabs. Gels were prepared containing a 5.0% concentration of acrylamide and a 3.5% concentration of the acrylamide monomer of the cross-linking agent methylene bisacrylamide. Ampholines in the ph ranges of 3-6 and were used at a concentration of 2.0% and polymerization of the gels was effected by ammonium persulfate in a final concentration of 0.035%. Serum samples of reference and patient sera were applied to filter paper tabs (3 M) 1 cm. by 0.5 cm., using approximately 15 xl. serum per tab. The cathode wick buffer was 0.4% ampholine, ph 5-8 (1 ml. per 100 ml. of 40% ampholine) and the anode wick buffer was 1.0 M phosphoric acid. Separations were carried out for 2 hours with increasing power settings, increased from an initial 2.5 watts for 30 minutes, 5 watts for the next 45 minutes, 12.5 watts for 30 minutes, and 16 watts for the final 15 minutes. The ph gradient was confirmed

3 734 ALLEN, HARLEY AND TALAMO A.J.C.P. Vol. 62 as being established when the 0.04% bromphenol blue dye added to the cathode buffer wick had focused as a sharp yellow line at the anode wick edge. This routinely occurred just prior to raising the power from 12.5 to 16 watts. The filter paper tabs were removed as soon as the albumin, marked by the tracking dye, had migrated out of the tabs, after one hour and 20 minutes. Following completion of focusing, the gels were removed, an edge portion removed, and sections cut at 1-mm. intervals from the edge strip for subsequent ph determination. The remainder of the slab was placed in 12.5% trichloroacetic acid for 10 minutes to fix the proteins. The gel was then washed in water and then placed in ethanol: acetic acid: coomassie blue stain, (45 ml. aqueous 0.2% coomassie brilliant blue, 45 ml. absolute ethanol, 10 ml. glacial acetic acid) and agitated for 10 minutes at 60 C. The stain was removed and the gel destained for 10 minutes in two 5-minute changes of destain solution consisting of 65 ml. H 2 0, 25 ml. absolute ethanol, 10 ml. glacial acetic acid. The gel was then transferred to 10% acetic acid and the patterns interpreted. Separations carried out over 24-cm. distances rather than the 11 cm. in the standard system were similarly handled, except that power settings were increased and focusing times were about 4'2 hours. Microdensitometry was performed on the stained gels employing an Ortec model 4300 integrating microdensitometer.* Initial localization and identification of the alpha-1-antitrypsin bands were accomplished by utilizing two-dimensional electrophoresis, employing a discontinuous buffer system at a constant ph in step gradient gels 2 and removing the albumin region of an MM phenotype. The albumin and alpha-1-antitrypsin contained in this band were then eluted and the pres- * Ortec, Inc., 100 Midland Road, Oak Ridge, Tennessee FIG. 1. A, Human Pi phenotype MM serum containing 5.81 gm. per 100 ml. protein, 1.25 mg. per ml. trypsin-inhibitory capacity, and 3.0 mg. per ml. alpha-1-antitrypsin by radial immunodiffusion separated by discontinuous electrophoresis at a constant ph in a % T 3.5% C step gradient acrylamide gel slab. The excised albumin-alpha-1-antitrypsin region is marked by the arrow. B, Albumin-alpha-1-antitrypsin eluate from A concentrated to a value of 2.22 mg. per 100 ml. protein containing 1.0a mg. per ml. trypsin-inhibitory capacity and 1.0 mg. per ml. alpha-1-antitrypsin by radial immunodiffusion, separated by isoelectric focusing. C, Same whole serum as in A separated side-by-side with eluate B. B

4 December 1974 PI PHENOTYPING BY ISOELECTRIC FOCUSING 735 JO' S I FZ MM MZ MZ ZZ ZZ ZZ MS MM? PH SL I FIG. 2. Various Pi phenotypes separated by isoelectric focusing on a ph 3-5 gradient acrylamide gel slab. The broken line indicates the ph gradient, which may be determined at each point by dropping a vertical line to the ph scale on the X axis. ence of alpha-1-antitrypsin confirmed by trypsin-inhibitory capacity and radial immunodiffusion. The resulting eluate was then separated in the second dimension by the isoelectric focusing described above. One-half of the gel was stained with coomassie brilliant blue and the other half reacted with overlayered alpha-1-antitrypsin antiserum. Results The localization of the alpha-1-antitrypsin components in the MM Pi phenotype is shown in Figure 1. The protein band shown in this region also showed corresponding precipitin lines, following reaction with alpha-1-antitrypsin antiserum. The albumin alpha-1- antitrypsin fraction contained protein bands similar in number and distribution to those found in the corresponding whole serum. The weak bands at the lower portion of the gel with an isoelectric point of about ph 4 were also present in the albumin fraction but were too faint to photograph. The Pi isoelectric focusing patterns of the alleles F, M, S, and Z are shown in Figure 2. The measured ph gradient is shown by the dashed line laid directly over the gel. With little experience these various phenotype patterns are readily recognized visually, although differentiation of MS and SS (not shown) is aided by microdensitometry. When run side-by-side, all bands can be referenced with respect to their distances from the first albumin zone (arrow) of a reference MM serum and precise location of each protein of its relative zone determined either by direct measurement or from the densitometric trace. Patterns may be further studied as illustrated in Figures 3 and 4. Densitometry shows a distribution of peaks similar to that obtained by acid starch gel followed by crossing antigenantibody electrophoresis. The resulting MM, MZ, and ZZ patterns compared with the same sample as separated by acid starch gel electrophoresis followed by crossing antigen-antibody electrophoresis are shown in the inset with each den-

5 736 ALLEN, HARLEY AND TALAMO AJ.C.P. Vol. 62 As may be observed, both visually and by microdensitometry, all Pi types studied had various concentrations of bands, apparently under control of the M allele, which may be in too low an amount or insufficiently separated to be observed on crossing antigen-antibody electrophoresis patterns. With the two MZ patterns in figures 3 FIG. 3. Densitometric traces of separations 2, 3, and 4 shown in Figure 2. Full scale equals 0.8 optical density units. Tracings of the corresponding crossing antigen-antibody electrophoretic patterns from the same serum are inserted with each densitometric trace. Small arrows mark reference band 6 of the MM phenotype; the large arrow indicates the vertically alligned albumin reference peaks. sitometric trace (Figure 3). It is evident that higher resolution is obtained by isoelectric focusing and that the greater sensitivity of the coomassie blue protein stain provides more detail for both qualitative and quantitative interpretation. FIG. 4. Densitometric traces of separations 1, 4 and 8, Figure 2, with albumin peak, large arrow vertically aligned. Full scale equals 0.8 optical density units.

6 December 1974 PI PHENOTYPING BY ISOELECTRIC FOCUSING 737 Z i ' ^"^^^ ^^"^^" 0' M8,t> FIG. 5. Isoelectric points and distribution of reference sera Pi types M, S, F, Z phenotypes with major allele products schematically presented. The approximate isoelectric point of each allele product may be determined by extending the zone to the dashed line and dropping a vertical line to the ph scale on the X axis. M7 M6 MS "S^^^-.' M M M4. '' s M M ^ O, ;* ^^ ^^ ^ MS z,' ' m ^ MZ f, 4.3 '- -1 i Q '' 11 ' FZ f= F and 4, better resolution, i.e., less background, can be seen in fresh sera (Fig. 4) than in the older sera stored for six months at -20 C (Fig. 3), although pattern typing was not affected. In figure 5 the approximate isoelectric points of the various allele products from the most cathodal Z to the anodal M4 band are shown. The isoelectric points of the various allele products extend over a ph range of approximately 0.33 units. Single zones, for example, M-6 zones (Fig. 2), have an isoelectric points of ph 4.54 ± when calculated from a single gel and 4.49 ± 0.05 based on a series of determinations utilizing two different ph gradient ranges. Two zones separated by as little as 0.3 mm., which corresponds to an isoelectric point difference of ph units in the 11-cm. system employed, may be readily resolved. When similar separations were carried out over 24 cm., two protein zones with isoelectric points differing by approximately ph units may be resolved. However, gels 11 cm. long appeared sufficient to identify the alleles tested on a ph gradient. Separations on ph 3-6 gradients provided similar patterns for phenotyping; however, the separation between bands was somewhat less. These equivalent results are not shown since this range of ampholine is now no longer available. Discussion To clarify why these two apparently unrelated methods produce similar qualitative distributions, an understanding of the electrochemistry involved in the separations is pertinent. The acid starch gel method of Fagerhol 6 utilizes a discontinuous buffer system with an initial gel buffer ph of 4.95 and a cathode buffer at 6.3; thus, at the concentrations employed,

7 738 ALLEN, HARLEY AND TALAMO AJ.C.P. Vol. 62 the running ph will be determined by the Kohlrausch regulating function, which by direct measurement is approximately ph 7.5. The net effect of such a system appears to be to effectively reduce the net charge of the larger albumin molecule to below that of the alpha-1-antitrypsin components so that they migrate anodally to the albumin in the moderately sieving starch medium, with the resultant resolution affected by their various isoelectric points. Fortuitously, the other alpha-1 globulins have sufficiently higher isoelectric points so as not to interfere or are not detected in either the initial acid gel or in the subsequent crossing antigen-antibody electrophoresis step. Two-dimensional electrophoresis with cellulose acetate in the first dimension and discontinuous electrophoresis in step gradient acrylamide gel in combination with isoelectric focusing in either a second or third dimension have shown that the A-l-AT components have lower isoelectric points than the other alpha-1 components, which focus cathodal to albumin. 11 Pi allele products thus focus anodally to the albumin, which provides the rationale for the similar distributions in the two technics. The increased resolution in the latter procedure is to be expected, since proteins are maximally resolved when focusing is completed, while in the former method they diffuse with time once they begin to unstack following the passage of the moving boundary. In addition, the small molecular weight, 45,000, of the Pi allele products allows the use of a moderately stiff sieving gel which, being more easily handled during staining procedures, may be cast in 1-mm. thickness to further enhance resolution. 3 Fagerhol 8 has described the genetics of the Pi system as consisting of a number of codominant alleles, each of which produces a complex pattern of eight protein zones on acid starch gel electrophoresis. Thus, a comparison of the M allele and Z allele products should show eight protein zones in each, with the Z allele zones showing decreased mobility to corresponding M allele zones. A similar analysis by isoelectric focusing should demonstrate a slightly higher isoelectric point in the Z allele zones compared with the M allele zones. Analysis of the various allele products shown in Figure 2 indicated, for example, that the major protein zone designated by Fagerhol Z-2 9 (located in reference to the first albumin peak from measurements made directly on the gel and on the densitometric traces) was closer to the albumin than M6, which corresponds to an isoelectric point of ph units (P < 0.01) higher than the M6 zone. On the other hand, the same 12 protein zones are apparent in the ZZ phenotype and the SS phenotype (not shown), rather than eight as expected. Similar analysis of these additional zones indicated correspondence in their isoelectric points to the M4, 5, 7, and 8 zones in the ZZ and M4, 5, and 7 in the SS phenotype, although they are greatly reduced in amount. The FZ (Fig. 2) reference phenotype serum, which demonstrated 16 protein zones, also contained four detectable protein zones with isoelectric points similar to M4, 5, 7, and 8. These components are not apparent, for example, in the ZZ pattern from acid starch gel electrophoresis, crossing antigen-antibody electrophoresis pattern (Fig. 3, insert). These observations, although on a limited number of Pi types, suggest that by isoelectric focusing M allele products may be detected in possibly all Pi types where to date they have been unsuspected. Further studies are required to ascertain whether these are true M allele proteins and to determine how many and in what amount they may be present. A number of protein zones with isoelectric points in the region of ph 4.0 which appear to differ from individual to individual are found. A similar region, which

8 December 1974 PI PHENOTYP1NG BY ISOELECTRIC FOCUSING 739 also has nonspecific esterase activity and is under direct genetic control, has been found in inbred mice 1 ; at present, the significance of this finding in human plasma has not been further studied. While only a few of the known Pi allele types have been studied here by isoelectric focusing, it would appear that this technic has resolving capacity sufficient to differentiate the other Pi allele products. The FZ phenotype indicates that some 16 proteins are present over the 0.33-pHunit range from the M-l band to the most cathodal Z band. More than 20 Pi allele products can be resolved in this region on a gel 11-cm. long. Microdensitometry further expands this potential, in that quantitative relationships may be obtained as well as qualitative ones. Pi patterns obtained by both the acid starch gel and isoelectric focusing indicate similarity in general, but the resolving powers differ dramatically and quantification is not possible by starch gel electrophoresis. The present preliminary study suggests that isoelectric focusing may provide a valuable additional tool with which to study both the qualitative and the quantitative relationships of the various Pi allele products and their relationship to diseases associated with antiproteinase deficiencies. Acknowledgment. Ms. Carol Langley and Ms. Mary Corrigan provided technical assistance. References 1. Allen RC: Separation and quantification of mouse plasma esterases by discontinuous electrophoresis and isoelectric focusing on polyacrylamide gel. U.S.P.H.S., Contract report # Pr , Nat Inst Environmental Health Sciences, Allen RC: Polyacrylamide gel electrophoresis with discontinuous voltage gradients at constant ph, Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel. Ed. by RC Allen, HR Maurer. Berlin, DeGruyter, 1974, pp Allen RC: Prerequisites for quantitative microdensitometry, Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel. Edited by RC Allen, HR Maurer. Berlin, DeGruyter, 1974, pp Bundy HE, Mehl JW: Trypsin inhibitors of human serum. II. Isolation of the a-\ inhibitor and its partial characterization. J Biol Chem 234: , Cohn EJ, Hughes WL, Weare JH: Preparation and properties of serum and plasma proteins. XIII. Crystallization of serum albumin from ethanol-water mixtures. J Am Chem Soc 69: , Fagerhol MK: Acid starch gel electrophoresis for detection of alpha-antitrypsin variants (Pi types): Outline of techniques employed currently, Pulmonary Emphysema and Proteolysis, Edited by C. Mittman. New York, Academic Press, 1972, pp Fagerhol MK: Quantitative studies on the inherited variants of serum alpha,-antitrypsin. Scand J Clin Lab Invest 23:97-102, Fagerhol MK: Genetics of the Pi system, Pulmonary Emphysema and Proteolysis. Edited by C Mittman. New York, Academic Press, 1972, pp Laurell C-B, Eriksson S: The electrophoretic alpha,- globulin pattern of serum in alpha,- antitrypsin deficiency. Scand J Clin Lab Invest 15: , Laurell F: Antigen-antibody crossed electrophoresis. Anal Biochem 10: , Maurer HR, Allen RC: Polyacrylamide electrophoresis in clinical chemistry: Problems of standardization and performance, recent advances and suggestions for optimizing fractionation. Clin Chim Acta 40: , Myerowitz RL, Chrambach A, Robard D, et al: Isolation and characterization of mouse serum alphaj-antitrypsins. Anal Biochem 48: , Talamo RC: Unpublished data