- Email: [email protected]
The preparation of serum albumin for turnover studies
COMMUNICATIONS The Preparation of Serum Albumin for Turnover Studies Proteins chemically labeled with I311 have been used extensively to estimate turnover rates in viva (1). Discrepancies among the reported results persist (2). Labeling of albumin with an a-aminoisobutyric acid-l-l% (AIB) derivative as an alternative to the 1311procedure has been proposed (3). In the present study rabbit and human albumin isolated by starch block electrophoresis (4) or ethanol-water fractionation (5) has been used. Native and labeled albumins were compared on the basis of immune response, electrophoretic migration, sedimentation velocity, and absorption spectrum. Activation of the ilIB and coupling of the product with albumin has been described previously (3). Minor modifications in the method involved the following: the amino acid-to-protein ratio was descreased (22.9 p~:l pi), the reaction mixture was centrifuged (30 min, 40,000 rpm) to eliminate turbidity, and exhaustive dialysis was instituted to assure the removal of unbound AIB derivative. Iodination, for the comparative study, was by astandard procedure (6). Concentrations of albumin were determined at 280 rn&, except for the “ponceau S-stained preparations which were determined at 520 mp. Based on a constant specific activity analysis, the ratio of AIB bound to albumin was 1:l. No qualitative differences among native and labeled (carbon or iodine) albumins were detected by disc gel electrophoresis. The several lots of ethanol-
FIG. 1. Acrylamide gel (7.570) patterns of native rabbit albumin isolated by starch block electrophoresis. The serum was obtained at a single bleeding from one animal and stored at -20”. Reading from left to right the albumin was isolated from serum stored 4 days, 14 days, and 330 days.
351
water isolated rabbit albumin had fewer bands than the human albumin. Disc gel electrophoresis of rabbit albumin isolated by starch block electrophoresis exhibited increasing numbers of bands in preparations obtained from serum stored at -20” longer than a week prior to isolation (Fig. 1). The change in number of bands was not attributable to serum differences among animals, nor to barbital salts used in the isolation, since both of these variables were maintained constant. Centrifugation and gel filtration effected no change in the multiple bands. All disc gel analyses were highly reproducible regardless of the source or concentration of protein used (0.25-1.50 mg/ml). The increased numbers of bands in the albumin isolated from stored serum possibly are indicative of hydrophobic interactions or shifts in disulfide cross-links during storage (7, 8). Apparently this type of change is not restricted to albumin. Hemoglobin has been reported to exhibit two bands if disc gel electrophoresis is performed within 4 to 5 days after collection, but if the sample is held longer than a week four bands develop; oxidation of the hemoglobin to methemoglobin with K3Fe(CN)G is without effect (9). Cellulose acetate paper electrophoresis of the W-labeled albumin compared with native albumin revealed that the labeled product migrated more rapidly toward the anode. This increased migration rate was also apparent in the immunoelectrophoresis patterns. Iodinated albumin also migrates more rapidly than does native albumin (1). The peak of W-radioactivity assumed to be bound to the albumin was found to migrate more rapidly toward the anode than did the peak of albumin (Fig. 2) and the spread (8 mm) between the two peaks was reproducible. High voltage paper electrophoresis of W-labeled albumin followed by scanning for radioactivity disclosed several radioactive areas, but only one area stained with bromophenol blue. The stained spot could be superimposed over the stain developed by native albumin run simultaneously. The nonstaining radioactive areas were ninhydrinnegative. Gel filtration (Sephadex G-25) effected no change in the radioactivity pattern, indicating that the nonstaining product most probably was not unbound AIB. Separation of staining and nonstaining radioactive products was accomplished with high voltage paper electrophoresis. Electrophoresis (high voltage paper) of the isolates and scanning for radioactivity showed that the products retained the migration velocities observed prior to their separation. Persistence of the association between the staining and nonstaining radioactive products further implies that variants of albumin molecules may produce the spread of radioactive substances (7, 8). Low concentrations
352
COMMUNICATIONS - 4000
32 ,.28 t
- 3000
2 .24 3 ; .20 z. .L_ t: .16-
f .I L -2000 x z : 8
z .; g .I2 5 e .06 .04
- low
t
0 -------- I 12.5
#’ I 50.0 25.0 37.5 Distancemigrated(mm)
62.5 t
FIO. 2. Distribution of rabbit albumin and l”C-label (assumed bound to the albumin) eluted from cellulose acetate paper after electrophoresis, staining, and elution. Verona1 buffer was used for both the electrophoresis and elution. The cellulose strip (120 X 24 mm) was cut crosswise (2 X 24 mm) for elution and the concentration was determined at 520 nq. Prior to the electrophoresis the 14Clabeled albumin was subjected to exhaustive dialysis, centrifugation, and DEAE-cellulose chromatography to remove unbound 14C. The specific activity of this labeled albumin was the same as that of the protein used to determine the ratio of AIB to protein.
REFERENCES 1. M&ARL.~NE, A. S., in “Mammalian Protein Metabolism, “Vol. I (“Metabolism of Plasma Proteins,” H. N. Munro and J. B. Allison, eds.), p. 298. Academic Press, New York (1964). 2. SCHULTZE, H. E., AND HEREMANS, J. F., in “Molecular Biology of Human Proteins,” Vol. I (“Turnover of the Plasma Proteins”), p. 450. Elsevier, Amsterdam (1966). 3. MARGEN, S., AND TARVER, H., in “Advances in Tracer Methodology,” Vol. II (“The Preparation of Labeled Albumin for Turnover Studies,” S. Rothchild, ed.), p. 69. Plenum Press, New York (1965). 4. KUNKEL, H. G., in “Methods of Biochemical Analysis,” Vol. I (“Zone Electrophoresis,” D. Glick, ed.), p. 155, Wiley, New York (1954). 5. Cohn, E. J., HUGHES, W. L., JR., AND WEARE, J. H., J. Am. Chem. Sot. 69, 1753 (1947). 6. GREENWOOD, F. C., HUNTER, W. M., AND GLOVER, J. S., &o&em. J. 89, 114 (1963). 7. ANDERSON, L., Biochim. Biophys. Acta 117, 115 (1966). 8. HARTLEY, R. W., JR., PETERSON, E. A., AND SOBER, H. A., Biochemistry 1, 60 (1960). 9. BROWN, S. J., Some biochemical differences in muscle from dystrophic and normal chickens, M. S. thesis, University of California, Berkeley (1967). MARION P. CULLEN HAROLD TARVER SHELDEN MARGEN Department of Nutritional Sciences
University of California of some of the detectable radioactive variants may explain failure to stain; however, the substance had a strong affinity for the AIB derivative, as the nonstained material was practically as highly labeled as that which stained. No differences in the immune response, sedimentation velocity, or absorption spectra of the native and labeled albumin were detected. Whether the %-amino acid-labeled albumin is more acceptable than 1311-labeled albumin in in viva turnover studies cannot be predicted because critical evaluation of either label will be hampered until the molecular variability of albumin is resolved. ACKNOWLEDGMENT One of the authors (M.P.C.) thanks Prof. R. David Cole, Department of Biochemistry, University of California, Berkeley, California, for his continuing interest and help in this research and in the preparation of this manuscript.
Berkeley, California 9.&7!% Received November 27, 1968; accepted April 5, 1969
Enzymic Degradation Fagopyrum vulgare
of Rutin in Leaves
The enzymic degradation of rutin in microorganisms and animals was found to yield protocatechuic acid and/or phloroglucinol (l-10). In higher plants, Ahlgrim (11) studied the change in rutin content in Fagopyrum vulgare and concluded that rutin was metabolized in higher plants. In this work, rutin was decomposed with crude enzyme from the leaf of Fagopyrum vulgare and the products were determined chromatographically. Leaves of different ages containing different amounts of rutin were also compared for the activity. The rutin decomposing-activity was found
FIG. 1. Acrylamide gel (7.570) patterns of native rabbit albumin isolated by starch block electrophoresis. The serum was obtained at a single bleeding from one animal and stored at -20”. Reading from left to right the albumin was isolated from serum stored 4 days, 14 days, and 330 days.
351
water isolated rabbit albumin had fewer bands than the human albumin. Disc gel electrophoresis of rabbit albumin isolated by starch block electrophoresis exhibited increasing numbers of bands in preparations obtained from serum stored at -20” longer than a week prior to isolation (Fig. 1). The change in number of bands was not attributable to serum differences among animals, nor to barbital salts used in the isolation, since both of these variables were maintained constant. Centrifugation and gel filtration effected no change in the multiple bands. All disc gel analyses were highly reproducible regardless of the source or concentration of protein used (0.25-1.50 mg/ml). The increased numbers of bands in the albumin isolated from stored serum possibly are indicative of hydrophobic interactions or shifts in disulfide cross-links during storage (7, 8). Apparently this type of change is not restricted to albumin. Hemoglobin has been reported to exhibit two bands if disc gel electrophoresis is performed within 4 to 5 days after collection, but if the sample is held longer than a week four bands develop; oxidation of the hemoglobin to methemoglobin with K3Fe(CN)G is without effect (9). Cellulose acetate paper electrophoresis of the W-labeled albumin compared with native albumin revealed that the labeled product migrated more rapidly toward the anode. This increased migration rate was also apparent in the immunoelectrophoresis patterns. Iodinated albumin also migrates more rapidly than does native albumin (1). The peak of W-radioactivity assumed to be bound to the albumin was found to migrate more rapidly toward the anode than did the peak of albumin (Fig. 2) and the spread (8 mm) between the two peaks was reproducible. High voltage paper electrophoresis of W-labeled albumin followed by scanning for radioactivity disclosed several radioactive areas, but only one area stained with bromophenol blue. The stained spot could be superimposed over the stain developed by native albumin run simultaneously. The nonstaining radioactive areas were ninhydrinnegative. Gel filtration (Sephadex G-25) effected no change in the radioactivity pattern, indicating that the nonstaining product most probably was not unbound AIB. Separation of staining and nonstaining radioactive products was accomplished with high voltage paper electrophoresis. Electrophoresis (high voltage paper) of the isolates and scanning for radioactivity showed that the products retained the migration velocities observed prior to their separation. Persistence of the association between the staining and nonstaining radioactive products further implies that variants of albumin molecules may produce the spread of radioactive substances (7, 8). Low concentrations
352
COMMUNICATIONS - 4000
32 ,.28 t
- 3000
2 .24 3 ; .20 z. .L_ t: .16-
f .I L -2000 x z : 8
z .; g .I2 5 e .06 .04
- low
t
0 -------- I 12.5
#’ I 50.0 25.0 37.5 Distancemigrated(mm)
62.5 t
FIO. 2. Distribution of rabbit albumin and l”C-label (assumed bound to the albumin) eluted from cellulose acetate paper after electrophoresis, staining, and elution. Verona1 buffer was used for both the electrophoresis and elution. The cellulose strip (120 X 24 mm) was cut crosswise (2 X 24 mm) for elution and the concentration was determined at 520 nq. Prior to the electrophoresis the 14Clabeled albumin was subjected to exhaustive dialysis, centrifugation, and DEAE-cellulose chromatography to remove unbound 14C. The specific activity of this labeled albumin was the same as that of the protein used to determine the ratio of AIB to protein.
REFERENCES 1. M&ARL.~NE, A. S., in “Mammalian Protein Metabolism, “Vol. I (“Metabolism of Plasma Proteins,” H. N. Munro and J. B. Allison, eds.), p. 298. Academic Press, New York (1964). 2. SCHULTZE, H. E., AND HEREMANS, J. F., in “Molecular Biology of Human Proteins,” Vol. I (“Turnover of the Plasma Proteins”), p. 450. Elsevier, Amsterdam (1966). 3. MARGEN, S., AND TARVER, H., in “Advances in Tracer Methodology,” Vol. II (“The Preparation of Labeled Albumin for Turnover Studies,” S. Rothchild, ed.), p. 69. Plenum Press, New York (1965). 4. KUNKEL, H. G., in “Methods of Biochemical Analysis,” Vol. I (“Zone Electrophoresis,” D. Glick, ed.), p. 155, Wiley, New York (1954). 5. Cohn, E. J., HUGHES, W. L., JR., AND WEARE, J. H., J. Am. Chem. Sot. 69, 1753 (1947). 6. GREENWOOD, F. C., HUNTER, W. M., AND GLOVER, J. S., &o&em. J. 89, 114 (1963). 7. ANDERSON, L., Biochim. Biophys. Acta 117, 115 (1966). 8. HARTLEY, R. W., JR., PETERSON, E. A., AND SOBER, H. A., Biochemistry 1, 60 (1960). 9. BROWN, S. J., Some biochemical differences in muscle from dystrophic and normal chickens, M. S. thesis, University of California, Berkeley (1967). MARION P. CULLEN HAROLD TARVER SHELDEN MARGEN Department of Nutritional Sciences
University of California of some of the detectable radioactive variants may explain failure to stain; however, the substance had a strong affinity for the AIB derivative, as the nonstained material was practically as highly labeled as that which stained. No differences in the immune response, sedimentation velocity, or absorption spectra of the native and labeled albumin were detected. Whether the %-amino acid-labeled albumin is more acceptable than 1311-labeled albumin in in viva turnover studies cannot be predicted because critical evaluation of either label will be hampered until the molecular variability of albumin is resolved. ACKNOWLEDGMENT One of the authors (M.P.C.) thanks Prof. R. David Cole, Department of Biochemistry, University of California, Berkeley, California, for his continuing interest and help in this research and in the preparation of this manuscript.
Berkeley, California 9.&7!% Received November 27, 1968; accepted April 5, 1969
Enzymic Degradation Fagopyrum vulgare
of Rutin in Leaves
The enzymic degradation of rutin in microorganisms and animals was found to yield protocatechuic acid and/or phloroglucinol (l-10). In higher plants, Ahlgrim (11) studied the change in rutin content in Fagopyrum vulgare and concluded that rutin was metabolized in higher plants. In this work, rutin was decomposed with crude enzyme from the leaf of Fagopyrum vulgare and the products were determined chromatographically. Leaves of different ages containing different amounts of rutin were also compared for the activity. The rutin decomposing-activity was found