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2. Gennari, C., Galli, M., and Montagnani, M., Urinary cyclic adenosine monophosphate in young adults and elderly subjects. J. Clin. Pat hot. 29,69 (1976). 3. Madsen, N. S.,Badawi, I., and Skovsted, L., A simple competitive protein-binding assay for adenosine-3’,5’-monophosphate in plasma and urine. Acta Endocri not. 81, 208 (1975). 4. Latner, A. L., and Pridhoe, K., A simplified competitive protein-binding assay for adenosine 3’,5’-monophosphate in plasma. Clin. Chim. Acta 48, 353 (1973). 5. Neelson, F. A., Drezner, M. K., Birch, B. M., and Lebovitz, H. A., Urinary cyclic adenosine monophosphate as an aid in the diagnosis of hyperparathyroidism. Lancet i, 631 (1973). 6. Schmidt-Gayk, H., and Roher, H. D., Urinary excretion of cyclic adenosinemonophosphate in the detection and diagnosis of primary hyperparathyroidism. Surg. Gynecot. Obstet. 137, 439 (1973). 7. Murad, F., and Pak, Ch. Y. C., Urinary excretion of adenosine 3’,5’-monophosphate and guanosine 3’,5’-monophosphate. N. Engi. J. Med. 286, 1382 (1972). 8. Taylor, A. L.,Davis, B. B., Pawlson, G. L., et al., Factors influencing the urinary excretion of 3’,5’-adenosine monophosphate in humans. J. Clin. Endocrinol. 30, 316 (1970). Harry Husdan Rudolph Vogi Dimitrios Abraham Departments of Medicine Clinical Biochemistry Toronto Western Hospital Oreopoulos Rapoport and and University of Toronto, Toronto, Ontario, Canada M5T 2S8 Amino Acid Sequence in Bovine Serum Albumin To the Editor: In this journal, Peters reviewed recent progress in the understanding of the structure and biosynthesis of albumin (1). Therein, the complete amino acid sequence of bovine serum albumin as reported by Brown (2) is reproduced. The sequences of nine peptides obtained during an investigation of tryptic peptides from bovine serum albumin enhancing the effect of bradykinin (3, 4) can generally be fitted into this sequence, but a few discrepancies exist. In the complete amino acid sequence of bovine serum albumin (2) the fragment Asp - Leu - Gly - Glu - Glu - HisPhe-Lys- (residues 13-20) is identical in amino acid composition with our peptide C-V, viz., Asp-(G1u2,Gly,Leu,Phe, His)-Lys. Because this peptide was formed by the action of trypsin, the Cterminal amino acid is lysine. The residue of a basic amino acid (lysine) at po- sition 12 also corresponds with the specific action of trypsin. We determined aspartic acidasthe N-terminal amino agreement with the proposed sequence of the fragment 180-184, as is also with theresidue ofa basicamino acidatpo- acid of peptide C-V. The isoelectric sition 179 (lysine). point which can be calculated (5) from fragment 13-20 (p1 4.2) fits with the mobilities of peptide C-V by high-voltage electrophoresis at pH 4.00 and 7.00. Therefore, as published, the aspartyl and glutamyl residues must be present as the free acids. The analytical results for this fragment are in full agreement with the results of Shearer et al. (6). The fragment -Leu-Val-Asn-GluLeu-Thr-Glu-Phe-Ala-Lys(residues 42-5 1) is identical in amino acid composition with our peptide B-Vu, viz., Leu-(Asx,Thr,Glx2,Ala,Val,Leu,Phe) Lys. Because this peptide was formed by the action of trypsin, the C-terminal amino acid is lysine. The residueof a basic amino acid at position 41 (lysine) also corresponds with the specific action of trypsin. We determined leucine as the N-terminal amino acid of peptide B-WI. The calculated isoelectric point of fragment 42-51 is p1 4.2. However, our peptide B-VII was almost neutral at pH 7.00.Therefore, only one of the three w-carboxylgroups can be free. This might be either the aspartyl residue or one of the two glutamyl residues. If the aspartyl residue 44 is an amide as published, then also one of the two giutamyl residues (45 or 48) must be amidated. In that case, the recalculated isoelectric point (p1 6.85) is in agreement with our results (p1 - 7.0). The fragment -Ala-Glu-Phe-ValGlu-Val-Thr-Lys(residues 224-231) is in amino acid composition identical with our peptide B-Ill, viz., Ala(Thr,Giu,Gln,Va12,Phe)-Lys. The residues of a basic amino acid at positions 223 and 231 (lysine) correspond with the specific action of trypsin. We determined alanine as the N-terminal amino acid of peptide B-Ill. The calculated isoelectric point of fragment 224-231 is p1 4.2. However, we found peptide B-Ill to be almost neutral at pH 7.00. Therefore, one of the two glutamyl residues present at positions 225 and 228 must be amidated. The published sequence of fragment 232-238, viz., -Leu-Val-Thr-Asp-LeuThr-Lys-, is in full agreement with our analytical results (amino acid composition, isoelectric point, N- and C-terminal amino acid) of peptide B-IV, viz., Leu-(Asp,Thr2,Val,Leu)-Lys. The same applies to the published sequence of fragment -Ala-Asp-Leu-Ala-Lyspeptide 256-260, viz., in relation to C-Il,viz., Ala-(Asp,Ala,Leu)- Lys. The residue of a basic amino acid (arginine) at position 225 is in agreement with our results. Because peptide C-IV enhances the effect of bradykinin, its amino acid sequence was determined by phenylisothiocyanide degradation as well as by The fragment -Thr-Cys-Val-Ala- massspectrometry. All our data (Thr- Asp-Glu-Ser-His-Ala-Gly-Cys-Glu-Lys(residues 52-64) is identical in amino acid composition with our peptide B-V, viz., Thr-(Asx, Ser, G1x2, Gly, Ala2, Cys2,Val,His)-Lys. Our results, namely the amino acid residues at position 51 Lys, 52 Thr, and 64 Lys, and a disulfide bridge in this fragment, agree with the published structure. The calculated isoelectric point of fragment 52-64 is p1 4.2. This value is lower than is to be expected from the mobilities of our peptide B-V on high-voltage electrophoresis at pH 4.00 and 7.00.Therefore, one of the carboxylgroups must be amidated. If residue 56 is Asn instead of Asp, the calculated isoelectric point (p1 5.4) of the modified fragment 52-64 corresponds with that of peptide B-V. One fragment of the published se- quence,viz., -Glu-lle-Ala-Arg- (residues 140-143) is in good agreement with our peptide D-IV: Glu-(Ala,Ile)-Arg. However, because D-IV is a tryptic peptide the amino acid residue in position 139 must be lysine or arginine instead of tyrosine as published. Because peptide D-V enhances the effect of bradykinin, its amino acid sequence was determined by phenylisothiocyanide degradation as well as by mass spectrometry. All our data, viz., Ile-Glu-Thr-Met-Arg, are in good Pro-Val-Ser-Glu-Lys) are in good agreement with the published sequence of fragment 464-469 except for the interchange of amino acid residues serine and glutamic acid at positions 467 and 468 in the published sequence. In my opinion our sequence is correct. Firstly, the sequence of peptide C-IV was determined by two different methods. Secondly, we have isolated a tryptic peptide from rabbit albumin enhancing the effect of bradykinin with the same sequence as C-IV and, finally, the sequence of the corresponding fragment (467-472) of human serum albumin is -Thr-Pro-Val-Ser-Asp-Lys(7), in which the serine is also followed by a dicarboxylic amino acid. In summary, the following corrections of the sequence determined by Brown are suggested: Residue 45 or 48 Gin instead of Glu, Residue 56 Asn instead of Asp, Residue 139 either stead of Tyr, Lys or Arg in- Residue 225 or 228 Gln instead of Glu, Residue 467 Ser instead of Glu and residue 468 Glu instead of Ser. References I. Peters, T., Jr., Serum albumin: Recent progress in the understanding of its structure CLINICALCHEMISTRY, Vol. 23, No. 7, 1977 1361 and biosynthesis. Clin. Chem. 23, 5 (1977). Review. 2. Brown, J. R., Structure of bovine serum albumin. Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 591 (1975). Abstract 2105. 3. Weijers, R. N. M., Thesis, University of Utrecht, The Netherlands, 1971. 4. Weijers, R., Hagel, P., Das, B. ., and van der Meer, C., Tryptic peptides from rabbit and bovine albumin enhancing the effect of bradykinin. Biochim. Biophys. Acta 279,331 (1972). 5. Segel, I. H., Biochemical Calculations, John Wiley & Sons, Inc., New York, N. Y., 1968, pp 139-149, 347-349. 6. Shearer, W. I., Bradshaw, R. A., Gurd, F. R. N., and Peters, T., Jr., The amino acid sequence and copper (11)-binding properties of peptide (1-24) of bovine serum albumin. J. Biol. Chem. 242, 5451 (1967). 7. Meloun, B., Mor#{225}vek, L,, and Kostka, V., Complete amino acid sequence of human serum albumin. FEBS Lett. 58, 134 (1975). R. N. M. Weijers Laboratory Onze Lieve Amsterdam, of Clinical Chemistry Voouwe Gasthuis The Netherlands tenninal cyanogen bromide peptides of bevine plasma albumin. Arch. Biochem. Riophys. 153,627 (1972). 3. Meloun, B., Moravek, L., and Kostka,V., Complete amino acid sequence of human serum albumin. FEBS Lett. 58, 134 (1975). 4. Spencer, E. M., Amino acid sequence of the alanyl peptide from cyanogen bromide cleavage plasma of bovine albumin. Arch. Biochem. Biophys. 165,80 (1974). 5. Brown, J. R., personal communication, March 1977. 6. Brown, J. R., personal communication, September The Mary Imogene (affiliated with 12 Peters, Jr. Bassett Hospital Enzyme Immunoassayof To the Editor: careful work in both laboratories. With regard to the minor corrections Dr. Weijers suggested for the sequence reported in this journal (1), only the last one is clearly indicated. 1. Glu at residues 45 and 48 and Asp at 56 were confirmed by King and Spencer (2). The sequence of human albumin is also in agreement (3). 2. Tyr at residue 139 was confirmed by Spencer (4) and by homology with termining phenytoin and phenobarbital in serum by adapting the EMIT system (Syva, Palo Alto, Calif. 94304) to the CentrifiChem (Union Carbide, Rye, N. Y. 10580). Both drugs can be assayed after one dilution of standards and samples. All reagents are prepared according to kit instructionsexcept as stated below. Controls: Syva-AED Control Procedure: Pipet 200 zl of calibrators and sample serum into 12 X 75 mm polypropylene tubes. To each of these add 400 Ml of EMIT AED Buffer(pH 7.9). The diluted specimens and standards are then transferred to specimen specificity, cleaves after Tyr13g. 3. Direct confirmation of the absence of amide residues at Glu2 and Glu2ss is cups for the pipettor. albumin lacking, but Dr. Brown notes that his peptide 224-23 1, corresponding to Dr. Weijers’ peptide B-Ill, was acidic rather than neutral (5). 4. Subsequent to his initial publication of the bovine albumin sequence Brown has agreed that residues 467-468 should be Ser-Glu rather than Glu-Ser (6), so that this correction should made in the sequence reported be (1). References 1. Peters, T., Jr.,Serum albumin: Recent progress in the understanding of its structure and biosynthesis. Clin. Chem. 23, 5 (1977). 2. King, T. P., and Spencer, E. M., Amino acid sequences of the amino and the carboxyl 1362 CLINICAL CHEMISTRY, n = n = lOs, T = = 0.25 8 for phenytoin 5 for phenobarbital The fmal printout is used for plotting. The zero standard absorbance value (zS.Ao)is subtracted from values of all other standards and samples before plotting. The net result (A &t0) is then plotted on log-log graph paper used in an Epilepsy Foundation of America, AED Quality Control Program (Dr. C. E. Pippenger, Columbia Presbyterian Medical Center, New York, N. Y. 10032), in which samples containing weighed-in quantities of drugs were sent to us for assay. Our results correlated well with established values for the 36 samples received. The coefficients of correlation were: phenytoin, 0.9937; phenobarbital, 0.9980. Morris London Dolores Sanabria David Yau Brookdale Hosp. Med. Center Brooklyn, N. 1’. 11212 Reagent: Syva-EMrr-Anti-epileptic Drug (AED) Dilantin assay kit Syva-EMIT-AED Phenobarbital assay kit Standards: Syva-AED Calibrators (3). Dr. Brown (6) found that trypsin, contrary to its usual human absorbance Besides the two quality controls in this study, we also participated We wish to describe a method for de- To the Editor: It is reassuring that the peptides isolated by Weijers in 1972 confirm in almost every respect the corresponding portions of the sequence of bovine albumin publishe4 independently by Dr. Brown in 1975. Such agreement bespeaks 340 nm, corresponding setting prqvided with the kit. N. Y. 13326 Phenytoin and Phenobarbftalwith the CentrIfugal Analyzer Dr. Peters responds: blank, Terminal, Operate Ab- Auto sorbance. - Columbia University) Cooperstown, Analyzer Setting: T0 16 1975. Theodore A buffer blank cup after the highest standard Specimensinduplicates The EMIT Reagent kit consists of Reagent A (Antibody/Substrate) and Reagent B (Enzyme). Pipet 20 il of Reagent A manually into the sample wells(topwell)of the transferdisc.Add 600 Ml of Reagent B to 9.9 ml of buffer and then transfer to the reagent dish on the pipettor. This volume will be sufficient for at least 20 samples. Pipettor setting: Reagent volume, 350 Sample volume, 10 Total volume, 50 Last sample plug Erroneously Low Creatine Klnase Activity Measurementswith Use of the Caiblochem CPK-MB Kit with the BeckmanTR EnzymeAnalyzer To the Editor: Recent reports indicate that the auto- mated Beckman Enzyme Activity Analyzer, system TR, may yield falsely negative results for high-activity samples because of substrate depletion (1, 2). We would like to report another distressing observation. The system TR in the automated mode reports lower activities than when run on the continuous mode at the lower end of the normal range for creatine kinase (EC 2.7.3.2). This is a particularly frequent occurrence when the Calbiochem CPK-MB kit is used without dithiothreitol activation to measure CPK-MM (3). Table 1 lists some typical findings with use of this kit. The average continuous-mode result is five times greater than the average automated mode result, a signifiThe cupson thetraycanbearranged cant difference. as follows: What Standards Vol. 23, No. 7, 1977 in duplicates causes this difference? probable cause is that the instrument The in