[9] Relating proteins by amino acid composition

Amino acid composition analysis is a classical protein analysis method, which finds a wide application in medical and food science research and is indispensable for protein quantification. It is a complex technique, comprising two steps, hydrolysis of the substrate and chromatographic separation and detection of the residues. A properly performed hydrolysis is a prerequisite of a successful analysis. The most significant developments of the technology in the last decade consist in the (i) reduction of the hydrolysis time by the use of microwave radiation energy; (ii) improvement in the sensitivity of the residue detection, the quantification of the sensitive residues and separation of the enantiomeric forms of the amino acids; (iii) application of amino acid analysis in the large-scale protein identification by database search; and (iv) gradual replacement of the original ion exchange residue separation by reversed-phase high-performance liquid chromatography. Amino acid analysis is currently facing an enormous competition in the determination of the identity of proteins and amino acid homologs by the essentially faster mass spectrometry techniques. The amino acid analysis technology needs further simplification and automation of the hydrolysis, chromatography and detection steps to withstand the pressure exerted by the other technologies.

Lipoprotein fractions in Rana esculenta were separated using the same salt intervals currently applied for human lipoproteins. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were analyzed with reference to the electrophoretic pattern. The lipoprotein electrophoretic pattern in males and females throughout the reproductive cycle showed minor differences. In general, each fraction was characterized by a specific apolipoprotein content. VLDL and LDL fractions were dominated by a high molecular weight (MW) band, most likely the counterpart of human Apolipoprotein B (apo B). The apo B in R. esculenta cross reacted, although weakly, with antibodies raised against chicken apo B. The HDL fraction showed a band with an apparent MW of 29 kDa. The electrophoretic mobility of the protein moiety of HDL was similar to human apolipoprotein A-I (apo A-I). However, HDL apolipoprotein of R. esculenta did not cross react with antibodies against chicken apo A-I under either denaturing or native conditions. The HDL apolipoprotein of R. esculenta was purified by DEAE-Sephacel chromatography followed by HPLC. Its amino acid composition showed a moderate correlation with trout, salmon, chicken and human apo A-I.

Macromolecular assemblages intimately associated with biogenic silica in plants are released on solubilization of the siliceous phase by treatment with buffered aqueous solutions of HF following treatment of plant material with concentrated oxidizing acids (perchloric, nitric and sulphuric) to remove cytoplasmic contents and the largely polysaccharidic cell wall. The non-dialysable material, which may form 0.015–0.030% dry weight of the silica, has an amino acid composition rich in Pro-Glu, Pro-Lys, or Ser-Asp-Gly depending on the extent of treatment with oxidizing acids. The materials are suggested to have an intrasilica location with materials of similar composition being extracted from the branches of Equisetum telmateia (a primitive plant) and from hairs from the lemma of the grass Phalaris canariensis. The role of such material in regulating nucleation, particle growth and aggregate structure development in silicas is discussed.

The riboflavin-binding protein, purified from goose egg yolk, is significantly larger than those previously isolated from other avian species. This is, in part, due to a larger polypeptide chain, but mainly to a much higher carbohydrate content, principally glucose and sialic acid. Approximately one-third of the glucose residues can be released by incubation with a mixture of exo-1,4-α- and exo-1,4-β-glucosidases. The amino acid composition is similar to that of other riboflavin-binding proteins, but the peptide maps are distinct from those of domestic fowl. The goose egg yolk protein has a much higher proportion of β-sheet than either domestic fowl or quail riboflavin-binding protein.

Lactate dehydrogenase (LDH) was purified from the echinoderm Holothuria atra. The enzyme occurs as a single L-specific homotetramer with a subunit molecular weight of 39,000, and kinetic properties that differ from those of the A₄, B₄ and C₄ enzymes of lower vertebrates and lower chordates. Amino acid compositional analysis and immunochemical titration indicate considerable structural affinity (⋍70% sequence similarity) with the single LDH subunit of the ascidan Pyura stolonifera. This supports the proposition that echinoderms lie close to the stemline leading from invertebrates to lower chordates, and that the ascidian LDH subunit represents the ancestral subunit type of vertebrate LDH.

Calmodulin was isolated and purified from shrimp abdominal muscle by heat precipitation, ion exchange and hydrophobic interaction chromatography. The purified calmodulin was homogeneous when evaluated by polyacrylamide gel electrophoresis. A still remaining contaminant was eliminated by high performance liquid chromatography on a phenyl column. The biological and physicochemical properties of shrimp calmodulin such as amino acid composition, molecular weight and the ability to activate calmodulin-deficient bovine heart phosphodiesterase were compared to those of other invertebrate calmodulins.