Proteins and Enzymes in Biological Evidence

Proteins are built from 20 standard amino acids. Each amino acid has the same backbone: a central alpha-carbon bonded to an amino group (NH2), a carboxyl group (COOH), a hydrogen, and a side chain that varies by amino acid. During protein synthesis, the carboxyl group of one amino acid forms a peptide bond with the amino group of the next, releasing water and creating a polypeptide chain. The sequence of amino acids in the chain, called the primary structure, is encoded in the gene and determines everything else about the protein.

Protein structure is organised at four levels. Primary structure is the amino acid sequence. Secondary structure refers to local regular folding patterns, principally alpha-helices and beta-sheets, stabilised by hydrogen bonds between backbone atoms. Tertiary structure is the overall three-dimensional fold of a single polypeptide chain, maintained by interactions between side chains: disulfide bonds, hydrogen bonds, ionic interactions, and hydrophobic packing. Quaternary structure applies to proteins composed of more than one polypeptide subunit; haemoglobin, for example, has four subunits.

Side chain chemistry governs how a protein interacts with its environment. Hydrophobic side chains cluster in the interior of a folded protein away from water. Charged and polar side chains face outward or line active sites where they interact with substrates or ligands. The physical chemistry of the R-groups explains why proteins are sensitive to pH: charged side chains gain or lose protons as pH shifts, altering the electrostatic balance that holds the fold together. This is why blood at extreme pH, as might occur in industrial accidents or chemical attack scenes, shows degraded protein markers even if the sample is fresh.

Enzymes accelerate reactions by binding their substrate at the active site, a precisely shaped pocket formed by specific amino acid residues, and stabilising the transition state of the reaction. The lock-and-key model describes substrate specificity as a geometric match between substrate and active site; the induced-fit model, now better supported experimentally, holds that the active site subtly changes shape upon substrate binding to achieve optimal catalytic geometry. Either way, the critical point is that enzyme activity depends on the integrity of the active site, and anything that disrupts the protein's three-dimensional shape reduces or eliminates catalytic activity.

Haemoglobin is not a true enzyme but has pseudoperoxidase activity because its haem iron centre (Fe in the ferric state) can accept electrons from a reducing substrate while hydrogen peroxide acts as the electron acceptor. The overall reaction oxidises the coloured indicator substrate, producing a colour change. Luminol (3-aminophthalhydrazide) emits blue chemiluminescence; reduced phenolphthalein (Kastle-Meyer reagent) turns pink; 3,3',5,5'-tetramethylbenzidine (TMB) turns blue. The reaction requires both hydrogen peroxide and the haem-containing protein; controls for each must be run alongside every casework test.

Sensitivity varies considerably between reagents. Luminol can detect blood diluted approximately one part in ten million, making it useful for scenes where blood has been cleaned, but this extreme sensitivity increases the false-positive risk. Phenolphthalein is sensitive to approximately one in a million. TMB is less sensitive but more specific. No single presumptive test is superior in all contexts: the choice depends on the substrate, the suspected level of dilution, and whether subsequent DNA extraction is planned. Luminol may not significantly inhibit subsequent PCR amplification at typical concentrations, but confirmatory tests should always be conducted on a separate portion of the sample where possible.

Presumptive tests indicate that blood may be present; they do not prove it. Confirmatory tests establish that the substance is blood, and species-specific confirmatory tests further establish whether it is human blood. The distinction matters in court: a positive luminol result on a cleaned floor is consistent with blood having been present, but a positive Takayama crystal test on a stain from the same location is evidence that a haemoglobin-derived crystal has formed, which is much harder to explain by coincidental contamination.

The Takayama (haemochromogen) test and the Teichmann (haemin) test are microcrystalline tests that exploit the chemistry of haemoglobin breakdown products. In the Takayama test, haemoglobin reacts with sodium hydroxide, glucose, and pyridine to produce characteristic salmon-pink pyridine-haemochromogen crystals. In the Teichmann test, haemoglobin is converted to haemin chloride crystals by acetic acid and chloride in the presence of heat. Both tests require microscopic examination to identify the crystal morphology. They are species-independent: they confirm haemoglobin but not human blood.

Immunochromatographic lateral-flow devices, sold commercially as HemaTrace (Seratec), ABAcard HemaTrace, and equivalent products, use antibodies specific to human haemoglobin (or human foetal haemoglobin) to confirm that a sample contains human blood. These devices work like pregnancy tests: a test line appears only if human haemoglobin is present. They are rapid, portable, and species-specific, making them the preferred confirmatory test in many laboratories. Cross-reactivity with primate blood is documented, so a positive result on a suspected wildlife crime scene requires further species typing. Courts in England and Wales, the United States, and India (under the Bharatiya Sakshya Adhiniyam 2023) all require confirmatory test evidence to establish that a stain is human blood before expert opinion on its significance is admitted.

When a biological sample is deposited at a scene, protein degradation begins immediately. The rate depends on temperature, humidity, UV exposure, pH, and the microbial load of the environment. Four mechanisms operate in most real-world samples: thermal denaturation, hydrolytic proteolysis, oxidative damage, and microbial enzymatic activity.

Thermal denaturation unfolds tertiary and secondary structure by disrupting non-covalent interactions. Most human proteins begin to unfold above about 40 to 50 degrees Celsius, and irreversible denaturation occurs progressively at higher temperatures. A bloodstain on a car dashboard in summer sunlight can reach 70 degrees Celsius within minutes. Denatured haemoglobin binds the luminol reaction substrate less efficiently, giving weaker or absent colour, and denatured antibodies in immunological assays fail to bind their target antigens. Heat also promotes Maillard reactions between amino groups and reducing sugars, producing cross-linked brown products that further alter protein structure.

Hydrolytic proteolysis is carried out by endogenous cellular proteases released when cells die (autolysis) and by proteases secreted by bacteria colonising the sample. These enzymes cleave peptide bonds, reducing proteins to small peptide fragments and eventually to free amino acids. In a moist environment at ambient temperature, significant proteolysis can occur within days. On a dry substrate, proteolysis is much slower because water is required for hydrolysis. The practical implication is that a bloodstain on a dry non-porous surface may retain detectable haemoglobin for months to years, while the same volume of blood on a moist porous substrate such as soil may be undetectable serologically within weeks.

Before DNA profiling became routine in the late 1980s and early 1990s, forensic serology relied on protein markers to characterise biological stains. ABO blood group typing was the earliest system, based on the presence or absence of glycoprotein antigens on red cell surfaces. These antigens are controlled by enzymes that add specific sugar residues to cell-surface proteins; the A and B alleles encode different glycosyltransferase enzymes, while the O allele encodes a non-functional enzyme, leaving the H antigen unmodified. ABO typing can still be performed on bloodstains using absorption-elution or agglutination-inhibition methods, and it remains useful when DNA is absent or degraded.

Isoenzyme typing extended individualisation beyond ABO. Enzymes such as phosphoglucomutase (PGM), esterase D, erythrocyte acid phosphatase (EAP), and adenylate kinase (AK) exist in variant forms across the human population. These variants, called isoenzymes or isozymes, differ in one or more amino acids, giving them slightly different electrophoretic mobility in a gel. A bloodstain extract run on a starch or polyacrylamide gel and stained with an enzyme-specific chromogenic substrate produces a banding pattern that reflects the individual's isoenzyme type. Combined across several systems, isoenzyme profiles could exclude most of the population as potential contributors.

Isoenzyme typing has been largely superseded by STR profiling, which is more discriminating, more sensitive, and more amenable to standardisation. However, isoenzyme methods remain relevant in three contexts: when samples are too old or degraded for reliable STR amplification; when the quantity of biological material is too small for current DNA extraction thresholds; and in jurisdictions or laboratories that have retained historical proficiency in the methods. Understanding isoenzyme typing also matters for interpreting older case records, particularly in cold cases where pre-DNA serology formed part of the original prosecution evidence.

Different biological evidence types present different protein profiles, and understanding these differences helps analysts select appropriate tests and interpret results. Blood is the richest source of forensic proteins: red cells contain haemoglobin and a range of isoenzymes; plasma contains albumin, immunoglobulins, clotting factors, and complement proteins. Semen contains prostate-specific antigen (PSA, also called p30), a serine protease produced by the prostate gland, which is the primary confirmatory marker for seminal fluid. Detection of PSA below nanogram-per-millilitre concentrations by immunochromatographic assay confirms semen regardless of azoospermia in the donor.

Saliva contains amylase, an enzyme that hydrolyses starch. Amylase activity is detectable in diluted saliva samples and is used as a presumptive marker for saliva. The starch-iodine test (Phadebas test) is widely used: a paper substrate impregnated with starch-dye conjugate turns blue-black in the absence of amylase and clears when amylase is present. Amylase is also present in low concentrations in other body fluids including vaginal secretions and blood, so a positive amylase result is a presumptive indicator for saliva rather than a confirmatory one. Species-specific immunological tests for human salivary amylase provide confirmation.

Bone, teeth, and hair present different protein challenges. The primary structural protein in bone is collagen (type I), which forms mineralised fibres in the bone matrix. Collagen is relatively resistant to degradation because it is tightly cross-linked and partially protected by the mineral phase, which is why ancient bone yields protein for analysis in some palaeontological and archaeological contexts. Forensic bone protein analysis, including collagen peptide mass fingerprinting by mass spectrometry, is an emerging method for species identification in wildlife crime investigations and for age estimation in human remains. The subject is covered in depth in Forensic Anthropology and wildlife applications in Wildlife Forensics.