Quantitative analyses of haemoglobin types in Australian horses.

The research article investigated the characteristics and ratios of types of haemoglobin, a protein found in horses’ blood, using specialized techniques like electrophoresis and isoelectric focusing. This involved examining haemoglobin variants at molecular level and their correlations to genetic attributes, and developing a method for accurately quantifying these variations.

Grouping Horses Based on Haemoglobin Type

• The initial classification of horses was done according to the type of haemoglobin (Hb) identified in their blood through a process called alkaline electrophoresis. This method separates differently charged particles, allowing identification of species of haemoglobin.
• Three groups emerged: a group with only a ‘fast component’ of haemoglobin, and two groups having both fast and slow components, but in differing ratios of 80:20 or 60:40.

The Role of Amino Acids in Haemoglobin Variants

• On a further level, these three groups of horses showed four different variants of the alpha-chain subunit of haemoglobin, traceable to two linked genes, identified by substitutions of certain amino acids – tyrosine for phenylalanine, and glutamine for lysine – at particular positions within the protein.
• This led to the definition of three haplotypes – distinct combinations of gene variants – labelled A, BI, and BII. So, the 100% ‘fast component’ group corresponded to ‘A’, and the 60:40 group to both ‘BI’ and ‘BII’. Horses in the 80:20 group exhibited either ‘A:BI’ or ‘A:BII’ haplotypes.

Methodologies Employed

• This research used isoelectric focusing, a technique to split protein variants based on their pH properties, which enabled a more detailed separation of the alpha-chain variants of haemoglobin.
• Due to the amino acid differences and the possible genetic recombination, up to 16 distinct genotypes could emerge. Identifying some of these required quantitatively analyzing patterns resulting from areas of differing concentrations of haemoglobin in the electrophoresis gel.
• Various methods were used to achieve this quantitative analysis, including scanning densitometry, a technique that measures the density of substances in gels or films, and spectrophotometry, which measures the amount of light absorbed by a substance.
• The article discussed other researchers’ use of laser scanning densitometry to quantify specific types of human haemoglobin, and laid out the authors’ own experiments using this method to quantify five types of equine haemoglobin, including two rare types.