Determination of immunoglobulin G in bovine colostrum and milk powders, and in dietary supplements of bovine origin by protein G affinity liquid chromatography: collaborative study.

An AOAC collaborative study was conducted to evaluate an affinity LC procedure for measuring immunoglobulin G (IgG) in selected dairy powders. The powders were extracted with 0.15 M sodium chloride solution and the pH was adjusted to 4.6 to precipitate caseins, which would otherwise lead to an overestimation of IgG. The analyte was then bound to a commercially available Protein G affinity cartridge and selectively eluted with a glycine buffer at pH 2.5. Detection was at 280 nm and quantification was made against a calibration curve prepared from bovine serum IgG. The samples analyzed included the likely matrixes for which this assay will find commercial use, namely, high- and low-protein-content colostrum powders, tablets containing colostrum powder, and some IgG-containing dairy powders; milk protein isolate, whey protein concentrate, and skim milk powder. Eleven laboratories provided data for the study and assayed blind duplicates of six materials. The repeatability RSD values ranged from 2.1 to 4.2% and the reproducibility RSD values ranged from 6.4 to 18.5%. The Protein G method with casein removal has adequate reproducibility for measuring IgG in colostrum-derived powders that are traded on the basis of IgG content as a colostral marker.

Validation of accuracy and precision of dual energy X-ray absorptiometry for infants.

There is limited information on the validity of bone mineral content measurement by dual energy X-ray absorptiometry (DXA BMC) for use in subjects with low body mass. We evaluated the accuracy and precision of DXA in piglets (body weight 886-5526 g, median 2096 g). Stepwise multiple regression analyses showed that ash weight is the major determinant of DXA BMC (adjusted r2 = 0.98, RMS residual = 3.61 g). The intercept was not significantly different from zero. DXA BMC measurements of other piglets under various clinical situations showed no significant effect from the use of cotton blanket, diaper, or positioning (prone, supine, lateral). In vivo replication of DXA BMC measurements of infants at a postnatal age of from 1-350 days showed a slope of 0.99 and high correlation coefficient (r2 = 0.99, RMS residual = 3.59 g). The intercept was not significantly different from zero, and the average coefficient of variation of duplicate DXA BMC in infants was 2.8%. We conclude that DXA BMC reliably but proportionately underestimates ash weight and is a highly precise method for measuring bone mineral status in young pediatric subjects.

High-performance affinity chromatography of DNA. II. Porosity effects.

A DNA-silica, (dT)18-silica, was prepared and used in a study of the chromatography of the oligonucleotide, (dA)18, based upon base pairing. It was shown that hybridization efficiency did not depend upon flow-rates up to 2 ml/min for the small columns (22 x 2 mm) used. As increasing amounts of (dA)18 were loaded onto the columns, the columns were found to saturate at a well defined capacity that was always less than the amount that theoretically could have been bound. Maximum capacity was achieved whenever the loading temperature was at least 20-25 degrees C below the temperature at which the loaded oligonucleotide would elute. The effects of porosity on both coupling efficiency and capacity were measured and suggest that pore sizes in the 300-500 A range are most appropriate for this form of chromatography.

Enzymatic syntheses of DNA-silicas using DNA polymerase.

Oligothymidylic acids couple to an activated ester silica (N-hydroxysuccinimidyl-silica) only when they contain an added aminoalkyl group. Heteropolymeric oligomers containing other nucleotide bases were shown to also couple by way of the nucleotide base (adenine, cytosine, or guanine); however, when a heteropolymeric oligonucleotide also contains a 5'-aminoalkyl moiety, coupling by way of the latter is the favored reaction. When duplex hybrids of oligonucleotides are formed, the nucleotide bases are protected from chemical coupling. Coupling by way of nucleotide bases would be detrimental to some chromatography experiments. On the basis of these observations, two different procedures were developed to produce DNA-silicas in which a single strand of the DNA is coupled by only its 5'-terminus. In the first of these, the polymerase chain reaction was used with a 5'-aminoalkyl primer to make a duplex DNA with one strand containing the 5'-aminoalkyl group and the duplex DNA is then coupled to the activated ester silica. This yielded a silica containing about 0.17 nmol of a 242-mer per gram silica which bound only probes specific for the coupled strand. In the other procedure, a template DNA strand was poly(A) tailed and hybridized to (dT)18-silica. DNA polymerase I (Klenow large fragments) was then used to copy the template-specified sequence directly onto the 3'-terminus of the (dT)18. This procedure yielded about 1.2 to 2.7 nmol DNA copied/g of silica of a specific 21-mer sequence. The DNA-silica produced selectively hybridized only with complementary sequences and not with DNA lacking that sequence. Either of these procedures thus produces DNA-silicas from heteropolymeric DNA sequences with a predetermined, specific 5'-terminal site of attachment.

Calmodulin specifically binds three proteins of the dystrophin-glycoprotein complex.

Dystrophin is the approximately 400,000 Da. protein (p400K) product of the Duchenne muscular dystrophy gene locus. In the sarcolemma membrane, it is associated with several other proteins, many of which are glycoproteins (abbreviated gp) and include gp156K, p59K, gp50K, gp43K, gp35K, and p25K. Here, we show that dystrophin, gp156K, and p59K are calmodulin-binding proteins, the binding is Ca(2+)-dependent, and of high-affinity similar to that seen with calmodulin-activated enzymes. Two putative calmodulin-binding sequences were identified, one at either end of the dystrophin sequence.

Trifluoperazine binding to mutant calmodulins.

Trifluoperazine (TFP) binding by 14 calmodulins, including 12 produced by site-directed mutagenesis, was determined. While vertebrate calmodulin binds 4.2 +/- 0.2 equiv of TFP, Escherichia coli expressed but unmutated calmodulins bind about 5.0 +/- 0.5 equiv of TFP. The cause for this difference is not known. The E. coli expressed proteins consist of two different series expressed from different calmodulin genes, CaMI and SYNCAM. The wild-type genes code for proteins that differ by nine conservative amino acid substitutions. Both these calmodulins bind 5 equiv of TFP with similar affinities, thus none of these conservative substitutions has any additional effect on TFP binding. Some altered calmodulins (deletion of EE83-84 or SEEE81-84, changing DEE118-120----KKK, M124----I,E120----K, or E82----K) have no appreciable effect on TFP binding. Other mutations affect either the binding of one TFP (deletion of E84) or about two TFP (changing E84----K, EEE82-84----KKK, E67----A, DEQ6-8----KKK, or E11----K). The mutations that affect TFP binding are localized to three regions of calmodulin: The amino-terminal alpha-helix, the central helix between the two globular ends of calmodulin, and a calcium-binding site in the second calcium-binding domain. The results are consistent with each of these regions either directly participating in drug binding or involved structurally in maintaining or inducing the correct conformation for TFP binding in the amino-terminal half of calmodulin.